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Sample records for receptor-mediated integrin-b1 activation

  1. Induction of aryl hydrocarbon receptor-mediated and estrogen receptor-mediated activities, and modulation of cell proliferation by dinaphthofurans.

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    Vondrácek, Jan; Chramostová, Katerina; Plísková, Martina; Bláha, Ludek; Brack, Werner; Kozubík, Alois; Machala, Miroslav

    2004-09-01

    A group of heterocyclic aromatic compounds, dinaphthofurans (DNFs), recently have been identified as potentially significant contaminants in freshwater sediments. In the present study, a battery of in vitro assays was used for detection of toxic effects of DNFs that are potentially associated with endocrine disruption and tumor promotion. Dinaphthofurans were found to act as relatively potent inducers of aryl hydrocarbon receptor (AhR)-mediated activity in the chemical-activated luciferase reporter gene expression DR-CALUX assay. The relative AhR-inducing potencies of DNFs were similar or even higher than relative potencies of unsubstituted polycyclic aromatic hydrocarbons (PAHs), with dinaphtho[1,2-b;2'3'-d]furan being the most potent AhR agonist. Two compounds, dinaphtho[2,1-b;2'3'-d]furan and dinaphtho[1,2-b;1'2'-d]furan, induced estrogen receptor (ER)-mediated activity in the estrogen receptor-mediated CALUX (the ER-CALUX) assay. Two types of potential tumor-promoting effects of DNFs were investigated, using in vitro bioassays for detection of inhibition of gap-junctional intercellular communication and detection of a release from contact inhibition. Although the acute inhibition of gap-junctional intercellular communication was not observed, all six tested DNFs were able to release rat liver epithelial WB-F344 cells from contact inhibition at concentrations as low as 100 nM. In summary, the present study indicated that DNFs can exert multiple biological effects in vitro, including induction of the AhR-mediated activity, release of cells from contact inhibition, and induction of ER-mediated activity.

  2. Lactate modulates the activity of primary cortical neurons through a receptor-mediated pathway.

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    Luigi Bozzo

    Full Text Available Lactate is increasingly described as an energy substrate of the brain. Beside this still debated metabolic role, lactate may have other effects on brain cells. Here, we describe lactate as a neuromodulator, able to influence the activity of cortical neurons. Neuronal excitability of mouse primary neurons was monitored by calcium imaging. When applied in conjunction with glucose, lactate induced a decrease in the spontaneous calcium spiking frequency of neurons. The effect was reversible and concentration dependent (IC50 ∼4.2 mM. To test whether lactate effects are dependent on energy metabolism, we applied the closely related substrate pyruvate (5 mM or switched to different glucose concentrations (0.5 or 10 mM. None of these conditions reproduced the effect of lactate. Recently, a Gi protein-coupled receptor for lactate called HCA1 has been introduced. To test if this receptor is implicated in the observed lactate sensitivity, we incubated cells with pertussis toxin (PTX an inhibitor of Gi-protein. PTX prevented the decrease of neuronal activity by L-lactate. Moreover 3,5-dyhydroxybenzoic acid, a specific agonist of the HCA1 receptor, mimicked the action of lactate. This study indicates that lactate operates a negative feedback on neuronal activity by a receptor-mediated mechanism, independent from its intracellular metabolism.

  3. Lactate modulates the activity of primary cortical neurons through a receptor-mediated pathway.

    Science.gov (United States)

    Bozzo, Luigi; Puyal, Julien; Chatton, Jean-Yves

    2013-01-01

    Lactate is increasingly described as an energy substrate of the brain. Beside this still debated metabolic role, lactate may have other effects on brain cells. Here, we describe lactate as a neuromodulator, able to influence the activity of cortical neurons. Neuronal excitability of mouse primary neurons was monitored by calcium imaging. When applied in conjunction with glucose, lactate induced a decrease in the spontaneous calcium spiking frequency of neurons. The effect was reversible and concentration dependent (IC50 ∼4.2 mM). To test whether lactate effects are dependent on energy metabolism, we applied the closely related substrate pyruvate (5 mM) or switched to different glucose concentrations (0.5 or 10 mM). None of these conditions reproduced the effect of lactate. Recently, a Gi protein-coupled receptor for lactate called HCA1 has been introduced. To test if this receptor is implicated in the observed lactate sensitivity, we incubated cells with pertussis toxin (PTX) an inhibitor of Gi-protein. PTX prevented the decrease of neuronal activity by L-lactate. Moreover 3,5-dyhydroxybenzoic acid, a specific agonist of the HCA1 receptor, mimicked the action of lactate. This study indicates that lactate operates a negative feedback on neuronal activity by a receptor-mediated mechanism, independent from its intracellular metabolism.

  4. Dopamine receptor-mediated mechanisms involved in the expression of learned activity of primate striatal neurons.

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    Watanabe, K; Kimura, M

    1998-05-01

    To understand the mechanisms by which basal ganglia neurons express acquired activities during and after behavioral learning, selective dopamine (DA) receptor antagonists were applied while recording the activity of striatal neurons in monkeys performing behavioral tasks. In experiment 1, a monkey was trained to associate a click sound with a drop of reward water. DA receptor antagonists were administered by micropressure using a stainless steel injection cannula (300 microm ID) through which a Teflon-coated tungsten wire for recording neuronal activity had been threaded. Responses to sound by tonically active neurons (TANs), a class of neurons in the primate striatum, were recorded through a tungsten wire electrode during the application of either D1- or D2-class DA receptor antagonists (total volume one of the surrounding barrels. SCH23390 (10 mM, pH 4.5) and (-)-sulpiride (10 mM, pH 4.5) were used. The effects of iontophoresis of both D1- and D2-class antagonists were examined in 40 TANs. Of 40 TANs from which recordings were made, responses were suppressed exclusively by the D2-class antagonist in 19 TANs, exclusively by the D1-class antagonist in 3 TANs, and by both D1- and D2-class antagonists in 7 TANs. When 0.9% NaCl, saline, was applied by pressure (<1 microl) or by iontophoresis (<30 nA) as a control, neither the background discharge rates nor the responses of TANs were significantly influenced. Background discharge rate of TANs was also not affected by D1- or D2-class antagonists applied by either micropressure injection or iontophoresis. It was concluded that the nigrostriatal DA system enables TANs to express learned activity primarily through D2-class and partly through D1-class receptor-mediated mechanisms in the striatum.

  5. EP3 receptors mediate PGE2-induced hypothalamic paraventricular nucleus excitation and sympathetic activation

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    Zhang, Zhi-Hua; Yu, Yang; Wei, Shun-Guang; Nakamura, Yoshiko; Nakamura, Kazuhiro

    2011-01-01

    Prostaglandin E2 (PGE2), an important mediator of the inflammatory response, acts centrally to elicit sympathetic excitation. PGE2 acts on at least four E-class prostanoid (EP) receptors known as EP1, EP2, EP3, and EP4. Since PGE2 production within the brain is ubiquitous, the different functions of PGE2 depend on the expression of these prostanoid receptors in specific brain areas. The type(s) and location(s) of the EP receptors that mediate sympathetic responses to central PGE2 remain unknown. We examined this question using PGE2, the relatively selective EP receptor agonists misoprostol and sulprostone, and the available selective antagonists for EP1, EP3, and EP4. In urethane-anesthetized rats, intracerebroventricular (ICV) administration of PGE2, sulprostone or misoprostol increased renal sympathetic nerve activity, blood pressure, and heart rate. These responses were significantly reduced by ICV pretreatment with the EP3 receptor antagonist; the EP1 and EP4 receptor antagonists had little or no effect. ICV PGE2 or misoprostol increased the discharge of neurons in the hypothalamic paraventricular nucleus (PVN). ICV misoprostol increased the c-Fos immunoreactivity of PVN neurons, an effect that was substantially reduced by the EP3 receptor antagonist. Real-time PCR detected EP3 receptor mRNA in PVN, and immunohistochemical studies revealed sparsely distributed EP3 receptors localized in GABAergic terminals and on a few PVN neurons. Direct bilateral PVN microinjections of PGE2 or sulprostone elicited sympathoexcitatory responses that were significantly reduced by the EP3 receptor antagonist. These data suggest that EP3 receptors mediate the central excitatory effects of PGE2 on PVN neurons and sympathetic discharge. PMID:21803943

  6. GRB2 Nucleates T Cell Receptor-Mediated LAT Clusters That Control PLC-γ1 Activation and Cytokine Production.

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    Bilal, Mahmood Yousif; Houtman, Jon C D

    2015-01-01

    GRB2 is a ubiquitously expressed adaptor protein required for signaling downstream of multiple receptors. To address the role of GRB2 in receptor-mediated signaling, the expression of GRB2 was suppressed in human CD4+ T cells and its role downstream of the T cell receptor (TCR) was examined. Interestingly, GRB2 deficient T cells had enhanced signaling from complexes containing the TCR. However, GRB2 deficient T cells had substantially reduced production of IL-2 and IFN-γ. This defect was attributed to diminished formation of linker for activation of T cells (LAT) signaling clusters, which resulted in reduced MAP kinase activation, calcium flux, and PLC-γ1 recruitment to LAT signaling clusters. Add back of wild-type GRB2, but not a novel N-terminal SH3 domain mutant, rescued LAT microcluster formation, calcium mobilization, and cytokine release, providing the first direct evidence that GRB2, and its ability to bind to SH3 domain ligands, is required for establishing LAT microclusters. Our data demonstrate that the ability of GRB2 to facilitate protein clusters is equally important in regulating TCR-mediated functions as its capacity to recruit effector proteins. This highlights that GRB2 regulates signaling downstream of adaptors and receptors by both recruiting effector proteins and regulating the formation of signaling complexes.

  7. Bovine lactoferrin counteracts Toll-like receptor mediated activation signals in antigen presenting cells.

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    Patrizia Puddu

    Full Text Available Lactoferrin (LF, a key element in mammalian immune system, plays pivotal roles in host defence against infection and excessive inflammation. Its protective effects range from direct antimicrobial activities against a large panel of microbes, including bacteria, viruses, fungi and parasites, to antinflammatory and anticancer activities. In this study, we show that monocyte-derived dendritic cells (MD-DCs generated in the presence of bovine LF (bLF fail to undergo activation by up-modulating CD83, co-stimulatory and major histocompatibility complex molecules, and cytokine/chemokine secretion. Moreover, these cells are weak activators of T cell proliferation and retain antigen uptake activity. Consistent with an impaired maturation, bLF-MD-DC primed T lymphocytes exhibit a functional unresponsiveness characterized by reduced expression of CD154 and impaired expression of IFN-γ and IL-2. The observed imunosuppressive effects correlate with an increased expression of molecules with negative regulatory functions (i.e. immunoglobulin-like transcript 3 and programmed death ligand 1, indoleamine 2,3-dioxygenase, and suppressor of cytokine signaling-3. Interestingly, bLF-MD-DCs produce IL-6 and exhibit constitutive signal transducer and activator of transcription 3 activation. Conversely, bLF exposure of already differentiated MD-DCs completely fails to induce IL-6, and partially inhibits Toll-like receptor (TLR agonist-induced activation. Cell-specific differences in bLF internalization likely account for the distinct response elicited by bLF in monocytes versus immature DCs, providing a mechanistic base for its multiple effects. These results indicate that bLF exerts a potent anti-inflammatory activity by skewing monocyte differentiation into DCs with impaired capacity to undergo activation and to promote Th1 responses. Overall, these bLF-mediated effects may represent a strategy to block excessive DC activation upon TLR-induced inflammation, adding

  8. CB1 receptor mediates the effects of glucocorticoids on AMPK activity in the hypothalamus.

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    Scerif, Miski; Füzesi, Tamás; Thomas, Julia D; Kola, Blerina; Grossman, Ashley B; Fekete, Csaba; Korbonits, Márta

    2013-10-01

    AMP-activated protein kinase (AMPK), a regulator of cellular and systemic energy homeostasis, can be influenced by several hormones. Tissue-specific alteration of AMPK activity by glucocorticoids may explain the increase in appetite, the accumulation of lipids in adipose tissues, and the detrimental cardiac effects of Cushing's syndrome. Endocannabinoids are known to mediate the effects of various hormones and to influence AMPK activity. Cannabinoids have central orexigenic and direct peripheral metabolic effects via the cannabinoid receptor type 1 (CB1). In our preliminary experiments, WT mice received implants of a corticosterone-containing pellet to establish a mouse model of Cushing's syndrome. Subsequently, WT and Cb1 (Cnr1)-knockout (CB1-KO) littermates were treated with corticosterone and AMPK activity in the hypothalamus, various adipose tissues, liver and cardiac tissue was measured. Corticosterone-treated CB1-KO mice showed a lack of weight gain and of increase in hypothalamic and hepatic AMPK activity. In adipose tissues, baseline AMPK activity was higher in CB1-KO mice, but a glucocorticoid-induced drop was observed, similar to that observed in WT mice. Cardiac AMPK levels were reduced in CB1-KO mice, but while WT mice showed significantly reduced AMPK activity following glucocorticoid treatment, CB1-KO mice showed a paradoxical increase. Our findings indicate the importance of the CB1 receptor in the central orexigenic effect of glucocorticoid-induced activation of hypothalamic AMPK activity. In the periphery adipose tissues, changes may occur independently of the CB1 receptor, but the receptor appears to alter the responsiveness of the liver and myocardial tissues to glucocorticoids. In conclusion, our data suggest that an intact cannabinoid pathway is required for the full metabolic effects of chronic glucocorticoid excess.

  9. Gamma-secretase activity of presenilin 1 regulates acetylcholine muscarinic receptor-mediated signal transduction

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    Popescu, Bogdan O; Cedazo-Minguez, Angel; Benedikz, Eirikur

    2004-01-01

    Familial Alzheimer's disease (FAD) presenilin 1 (PS1) mutations give enhanced calcium responses upon different stimuli, attenuated capacitative calcium entry, an increased sensitivity of cells to undergo apoptosis, and increased gamma-secretase activity. We previously showed that the FAD mutation...... causing an exon 9 deletion in PS1 results in enhanced basal phospholipase C (PLC) activity (Cedazo-Minguez, A., Popescu, B. O., Ankarcrona, M., Nishimura, T., and Cowburn, R. F. (2002) J. Biol. Chem. 277, 36646-36655). To further elucidate the mechanisms by which PS1 interferes with PLC-calcium signaling...... or PS1 D385N dominant negative cells. Our findings suggest that PS1 can regulate PLC activity and that this function is gamma-secretase activity-dependent....

  10. Estrogen receptor- and aryl hydrocarbon receptor- mediated activities of a coal-tar creosote

    Energy Technology Data Exchange (ETDEWEB)

    Fielden, M.R.; Wu, Z.F.; Sinal, C.J.; Jury, H.H.; Bend, J.R.; Hammond, G.L.; Zacharewski, T.R. [Michigan State University, East Lansing, MI (USA). Dept. of Biochemistry

    2000-05-01

    A coal-tar creosote was examined for estrogen receptor (ER)- and aryl hydrocarbon receptor (AhR)-mediated activity using a battery of mechanistically based assays. In vitro, creosote was found to bind the mouse ER, bind to the human sex hormone-binding globulin, and elicit partial agonist activity in reporter gene assays in transiently transfected MCF-7 cells. Based on competitive binding to the mouse ER, creosote contains approximately 165 mg/L of estradiol- equivalents. Creosote effectively transformed the AhR in vitro and induced a Cyp 1a1-regulated luciferase reporter gene in transiently transfected Hepa 1c1c7 cells. Based on dose-response curves, creosote contains approximately 730 mg/L of dioxin-equivalents. Creosote did not exhibit any AhR-mediated antiestrogenic activity in vitro. In vivo, creosote significantly induced liver pentoxyresorufin O- depentylation and ethoxyresorufin-O-deethylation (EROD) in a dose-dependent manner in ovariectomized (OVX) ICR mice, but did not increase uterine weight wet or vaginal cornification, due possibly to AhR-mediated antiestrogenic activity. In OVX DBA/2 mice, a strain less responsive to AhR ligands, creosote induced liver EROD to a lesser extent, but still did not show an increase in uterine wet weight or vaginal cornification. These results demonstrate that coal- tar creosote exhibits AhR- and ER-mediated activity in vitro, but its dioxinlike activity may suppress estrogenic response in vivo.

  11. Estrogen receptor- and aryl hydrocarbon receptor-mediated activities of a coal-tar creosote

    Energy Technology Data Exchange (ETDEWEB)

    Fielden, M.R.; Wu, Z.F.; Sinal, C.J.; Jury, H.H.; Bend, J.R.; Hammond, G.L.; Zacharewski, T.R.

    2000-05-01

    A coal-tar creosote was examined for estrogen receptor (ER)- and aryl hydrocarbon receptor (AhR)-mediated activity using a battery of mechanistically based assays. In vitro, creosote was found to bind to the mouse ER, bind to the human sex hormone-binding globulin, and elicit partial agonist activity in reporter gene assays in transiently transfected MCF-7 cells. Based on competitive binding to the mouse ER, creosote contains approximately 165 mg/L of estradiol-equivalents. Creosote effectively transformed the AhR in vitro and induced a Cyplal-regulated luciferase reporter gene in transiently transfected Hepa 1c1c7 cells. Based on dose-response curves, creosote contains approximately 730 mg/L of dioxin-equivalents. Creosote did not exhibit any AhR-mediated antiestrogenic activity in vitro. In vivo, creosote significantly induced liver pentoxyresorufin O-depentylation and ethoxyresorufin-O-deethylation (EROD) in a dose-dependent manner in ovariectomized (OVX) ICR mice, but did not increase uterine weight wet or vaginal cornification, due possibly to AhR-mediated antiestrogenic activity. In OVX DBA/2 mice, a strain less responsive to AhR ligands, creosote induced liver EROD to a lesser extent, but still did not show an increase in uterine wet weight or vaginal cornification. These results demonstrate that coal-tar creosote exhibits AhR- and ER-mediated activity in vitro, but its dioxinlike activity may suppress estrogenic responses in vivo.

  12. Tissue plasminogen activator inhibits NMDA-receptor-mediated increases in calcium levels in cultured hippocampal neurons

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    Samuel D Robinson

    2015-10-01

    Full Text Available NMDA receptors (NMDARs play a critical role in neurotransmission, acting as essential mediators of many forms of synaptic plasticity, and also modulating aspects of development, synaptic transmission and cell death. NMDAR-induced responses are dependent on a range of factors including subunit composition and receptor location. Tissue-type plasminogen activator (tPA is a serine protease that has been reported to interact with NMDARs and modulate NMDAR activity. In this study we report that tPA inhibits NMDAR-mediated changes in intracellular calcium levels in cultures of primary hippocampal neurons stimulated by low (5 μM but not high (50 μM concentrations of NMDA. tPA also inhibited changes in calcium levels stimulated by presynaptic release of glutamate following treatment with bicucculine/4-AP. Inhibition was dependent on the proteolytic activity of tPA but was unaffected by α2-antiplasmin, an inhibitor of the tPA substrate plasmin, and RAP, a pan-ligand blocker of the low-density lipoprotein receptor, two proteins previously reported to modulate NMDAR activity. These findings suggest that tPA can modulate changes in intracellular calcium levels in a subset of NMDARs expressed in cultured embryonic hippocampal neurons through a mechanism that involves the proteolytic activity of tPA and synaptic NMDARs.

  13. NR2D-containing NMDA receptors mediate tissue plasminogen activator-promoted neuronal excitotoxicity.

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    Baron, A; Montagne, A; Cassé, F; Launay, S; Maubert, E; Ali, C; Vivien, D

    2010-05-01

    Although the molecular bases of its actions remain debated, tissue-type plasminogen activator (tPA) is a paradoxical brain protease, as it favours some learning/memory processes, but increases excitotoxic neuronal death. Here, we show that, in cultured cortical neurons, tPA selectively promotes NR2D-containing N-methyl-D-aspartate receptor (NMDAR)-dependent activation. We show that tPA-mediated signalling and neurotoxicity through the NMDAR are blocked by co-application of an NR2D antagonist (phenanthrene derivative (2S(*), 3R(*))-1-(phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid, PPDA) or knockdown of neuronal NR2D expression. In sharp contrast with cortical neurons, hippocampal neurons do not exhibit NR2D both in vitro and in vivo and are consequently resistant to tPA-promoted NMDAR-mediated neurotoxicity. Moreover, we have shown that activation of synaptic NMDAR prevents further tPA-dependent NMDAR-mediated neurotoxicity and sensitivity to PPDA. This study shows that the earlier described pro-neurotoxic effect of tPA is mediated by NR2D-containing NMDAR-dependent extracellular signal-regulated kinase activation, a deleterious effect prevented by synaptic pre-activation.

  14. Adaptive and innate immune reactions regulating mast cell activation: from receptor-mediated signaling to responses

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    Tkaczyk, Christine; Jensen, Bettina M; Iwaki, Shoko

    2006-01-01

    In this article, we have described studies that have demonstrated that mast cells can be activated as a consequence of adaptive and innate immune reactions and that these responses can be modified by ligands for other receptors expressed on the surface of mast cells. These various stimuli...

  15. Estrogen receptor-mediated transcription involves the activation of multiple kinase pathways in neuroblastoma cells.

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    Clark, Sara; Rainville, Jennifer; Zhao, Xing; Katzenellenbogen, Benita S; Pfaff, Donald; Vasudevan, Nandini

    2014-01-01

    While many physiological effects of estrogens (E) are due to regulation of gene transcription by liganded estrogen receptors (ERs), several effects are also mediated, at least in part, by rapid non-genomic actions of E. Though the relative importance of rapid versus genomic effects in the central nervous system is controversial, we showed previously that membrane-limited effects of E, initiated by an estradiol bovine serum albumin conjugate (E2-BSA), could potentiate transcriptional effects of 17β-estradiol from an estrogen response element (ERE)-reporter in neuroblastoma cells. Here, using specific inhibitors and activators in a pharmacological approach, we show that activation of phosphatidylinositol-3-phosphate kinase (PI3K) and mitogen activated protein kinase (MAPK) pathways, dependent on a Gαq coupled receptor signaling are important in this transcriptional potentiation. We further demonstrate, using ERα phospho-deficient mutants, that E2-BSA mediated phosphorylation of ERα is one mechanism to potentiate transcription from an ERE reporter construct. This study provides a possible mechanism by which signaling from the membrane is coupled to transcription in the nucleus, providing an integrated view of hormone signaling in the brain.

  16. Endothelin potentiates TRPV1 via ETA receptor-mediated activation of protein kinase C

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    Furkert Jens

    2007-11-01

    Full Text Available Abstract Background Endothelin-1 (ET-1 both stimulates nociceptors and sensitizes them to noxious stimuli, an effect probably mediated by the ETA receptor (ETAR expressed in sensory neurons. The cellular mechanisms of this ET-1-mediated effect are only poorly understood. TRPV1, the heat-, pH- and capsaicin-sensitive cation channel already known to be modulated by a number of cellular mediators released in response to noxious stimuli and during inflammation, is a potential target for the action of ET-1. Results We studied the effects of ET-1 on TRPV1 in sensory neurons from the dorsal root ganglion (DRG and in HEK293 cells coexpressing TRPV1 and the ETAR. Specific 125I-ET-1 binding sites (817 ± 92 fmol/mg were detected in membrane preparations of DRG with an ETAR/ETBR ratio of 60:40. In an immunofluorescence analysis, coexpression of TRPV1 and the ETAR was found in a subpopulation of primary sensory neurons. ET-1 strongly potentiated capsaicin-induced TRPV1 currents in some neurons, and in HEK293 cells co-expressing TRPV1 and the ETAR. Weaker potentiation was observed in HEK293 cells coexpressing TRPV1 and the ETBR. ETAR activation also increased responses to low pH and heat. In HEK293 cells, strong potentiation of TRPV1 like that induced by ET-1 via the ETAR could be induced by PKC activation, but not with activators of the adenylyl cyclase or the PKA pathway. Furthermore, inhibition of PKC with bisindolylmaleimide X (BIM X or mutation of the PKC phosphorylation site S800 completely prevented ETAR-mediated potentiation. Conclusion We conclude that ET-1 potentiates TRPV1 by a PKC-dependent mechanism and that this could play a major role in the algogenic and hyperalgesic effects of ET-1 described in previous studies.

  17. Rapidly activated epidermal growth factor receptor mediates lipopolysaccharide-triggered migration of microglia.

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    Qu, Wen-Sheng; Liu, Jun-Li; Li, Chun-Yu; Li, Xiao; Xie, Min-Jie; Wang, Wei; Tian, Dai-Shi

    2015-11-01

    Previous reports have suggested that epidermal growth factor receptor (EGFR) is involved in microglia activation characterized by cell morphology changes, cytokine production and cell migration; and the biochemical regulation of the microglia migration is a potential therapeutic target following CNS inflammatory damages. However, the role of EGFR in microglia motility after inflammatory stimulation remains unknown. In the present study, lipopolysaccharide (LPS) was found to trigger rapid EGFR phosphorylation within 10 min, which was sustained during long-term stimulation in both primary microglial cells and the cultured BV2 microglial cells, furthermore, blocking EGFR phosphorylation by AG1478 significantly attenuated the LPS-induced chemotactic and chemokinetic migration of microglia. In addition, LPS could initiate calcium oscillation in microglia during live-cell recording, however, an intracellular calcium chelator and a selective inhibitor of calcium/calmodulin-dependent protein kinase II, but not an extracellular calcium chelator, remarkably suppressed the LPS-induced EGFR phosphorylation in BV2 microglia cells. As EGFR is not a traditional receptor for LPS, these findings suggest that the rapid phosphorylation of EGFR is attributed to the LPS-triggered intracellular calcium mobilization. By examining the downstream signals of EGFR, we further proved that extracellular signal-regulated kinase (ERK) is essential for EGFR-mediated microglia migration, because ERK inhibition attenuated the chemotactic and chemokinetic migration of microglia that had been induced by either LPS or EGF. Collectively, these results suggest that LPS could trigger the rapid phosphorylation of EGFR and subsequent ERK activation through mobilizing calcium activity, which underlies the microglia migration in an inflammatory condition.

  18. Valerian inhibits rat hepatocarcinogenesis by activating GABA(A receptor-mediated signaling.

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    Anna Kakehashi

    Full Text Available Valerian is widely used as a traditional medicine to improve the quality of sleep due to interaction of several active components with the γ-aminobutyric acid (GABA A receptor (GABA(AR system. Recently, activation of GABA signaling in stem cells has been reported to suppress cell cycle progression in vivo. Furthermore, possible inhibitory effects of GABA(AR agonists on hepatocarcinogenesis have been reported. The present study was performed to investigate modulating effects of Valerian on hepatocarcinogenesis using a medium-term rat liver bioassay. Male F344 rats were treated with one of the most powerful Valerian species (Valeriana sitchensis at doses of 0, 50, 500 and 5000 ppm in their drinking water after initiation of hepatocarcinogenesis with diethylnitrosamine (DEN. Formation of glutathione S-transferase placental form positive (GST-P(+ foci was significantly inhibited by Valerian at all applied doses compared with DEN initiation control rats. Generation of 8-hydroxy-2'-deoxyguanosine in the rat liver was significantly suppressed by all doses of Valerian, likely due to suppression of Nrf2, CYP7A1 and induction of catalase expression. Cell proliferation was significantly inhibited, while apoptosis was induced in areas of GST-P(+ foci of Valerian groups associated with suppression of c-myc, Mafb, cyclin D1 and induction of p21(Waf1/Cip1, p53 and Bax mRNA expression. Interestingly, expression of the GABA(AR alpha 1 subunit was observed in GST-P(+ foci of DEN control rats, with significant elevation associated with Valerian treatment. These results indicate that Valerian exhibits inhibitory effects on rat hepatocarcinogenesis by inhibiting oxidative DNA damage, suppressing cell proliferation and inducing apoptosis in GST-P(+ foci by activating GABA(AR-mediated signaling.

  19. Activation of Brain Somatostatin Signaling Suppresses CRF Receptor-Mediated Stress Response

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    Andreas Stengel

    2017-04-01

    Full Text Available Corticotropin-releasing factor (CRF is the hallmark brain peptide triggering the response to stress and mediates—in addition to the stimulation of the hypothalamus-pituitary-adrenal (HPA axis—other hormonal, behavioral, autonomic and visceral components. Earlier reports indicate that somatostatin-28 injected intracerebroventricularly counteracts the acute stress-induced ACTH and catecholamine release. Mounting evidence now supports that activation of brain somatostatin signaling exerts a broader anti-stress effect by blunting the endocrine, autonomic, behavioral (with a focus on food intake and visceral gastrointestinal motor responses through the involvement of distinct somatostatin receptor subtypes.

  20. P2X receptors mediate ATP-induced primary nociceptive neurone activation.

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    Bland-Ward, P A; Humphrey, P P

    2000-07-01

    ATP-gated P2X ion-channel receptors are localised throughout the mammalian nervous system and have been identified on neurones which participate in conduction of nociceptive information from the periphery to, and within, the CNS. This article briefly reviews recently published research describing the role that ATP and P2X receptors may play in pain perception, highlighting the importance of the P2X(3) receptor in this process. The P2X(3) receptor subunit is almost exclusively expressed on a subset of small and medium diameter sensory neurones innervating cutaneous and visceral tissue. Activation of P2X receptors present on the peripheral terminals of primary afferents results in neuronal depolarisation and, in conscious animals, leads to the manifestation of acute nociceptive behaviour. Recent animal studies have also shown that P2X(3) receptor expression is increased in sensory ganglia following acute neuronal injury, hinting that similar plasticity in the expression of this receptor subtype could underlie the mechanisms involved in a range of conditions characterised by sensory hypersensitivity in man. It is apparent from the evidence available that functional antagonists at specific P2X receptor subtypes could represent an important class of novel analgesic agents.

  1. P2X7 receptor mediates activation of microglial cells in prostate of chemically irritated rats

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    Heng Zhang

    2013-04-01

    Full Text Available Purpose Evidence shows that adenosine triphosphate (ATP is involved in the transmission of multiple chronic pain via P2X7 receptor. This study was to investigate the P2X7 and microglial cells in the chronic prostatitis pain. Materials and Methods Rats were divided into control group and chronic prostatitis group (n = 24 per group. A chronic prostatitis animal model was established by injecting complete Freund's adjuvant (CFA to the prostate of rats, and the thermal withdrawal latency (TWL was detected on days 0, 4, 12 and 24 (n = 6 at each time point in each group. Animals were sacrificed and the pathological examination of the prostate, detection of mRNA expression of P2X7 and ionized calcium binding adaptor molecule 1 (IBA-1 and measurement of content of tumor necrosis factor-α (TNF-α and interleukin-1β (IL-1β in the dorsal horn of L5-S2 spinal cord were performed on days 0, 4, 12 and 24. In addition, the content of TNF-α and IL-1β in the dorsal horn of L5-S2 spinal cord was measured after intrathecal injection of inhibitors of microglial cells and/or P2X7 for 5 days. Results The chronic prostatitis was confirmed by pathological examination. The expression of P2X7 and IBA-1 and the content of TNF-α and IL-1β in rats with chronic prostatitis were significantly higher than those in the control group. On day 4, the expressions of pro-inflammatory cytokines became to increase, reaching a maximal level on day 12 and started to reduce on day 24, but remained higher than that in the control group. Following suppression of microglial cells and P2X7 receptor, the secretion of TNF-α and IL-1β was markedly reduced. Conclusion In chronic prostatitis pain, the microglial cells and P2X7 receptor are activated resulting in the increased expression of TNF-α and IL-1β in the L5-S2 spinal cord, which might attribute to the maintenance and intensification of pain in chronic prostatitis.

  2. Fc Receptor-Mediated Activities of Env-Specific Human Monoclonal Antibodies Generated from Volunteers Receiving the DNA Prime-Protein Boost HIV Vaccine DP6-001.

    Science.gov (United States)

    Costa, Matthew R; Pollara, Justin; Edwards, Regina Whitney; Seaman, Michael S; Gorny, Miroslaw K; Montefiori, David C; Liao, Hua-Xin; Ferrari, Guido; Lu, Shan; Wang, Shixia

    2016-11-15

    HIV-1 is able to elicit broadly potent neutralizing antibodies in a very small subset of individuals only after several years of infection, and therefore, vaccines that elicit these types of antibodies have been difficult to design. The RV144 trial showed that moderate protection is possible and that this protection may correlate with antibody-dependent cellular cytotoxicity (ADCC) activity. Our previous studies demonstrated that in an HIV vaccine phase I trial, the DP6-001 trial, a polyvalent Env DNA prime-protein boost formulation could elicit potent and broadly reactive, gp120-specific antibodies with positive neutralization activities. Here we report on the production and analysis of HIV-1 Env-specific human monoclonal antibodies (hMAbs) isolated from vaccinees in the DP6-001 trial. For this initial report, 13 hMAbs from four vaccinees in the DP6-001 trial showed broad binding to gp120 proteins of diverse subtypes both autologous and heterologous to vaccine immunogens. Equally cross-reactive Fc receptor-mediated functional activities, including ADCC and antibody-dependent cellular phagocytosis (ADCP) activities, were present with both immune sera and isolated MAbs, confirming the induction of nonneutralizing functional hMAbs by the DNA prime-protein boost vaccination. Elicitation of broadly reactive hMAbs by vaccination in healthy human volunteers confirms the value of the polyvalent formulation in this HIV vaccine design. The roles of Fc receptor-mediated protective antibody responses are gaining more attention due to their potential contribution to the low-level protection against HIV-1 infection that they provided in the RV144 trial. At the same time, information about hMabs from other human HIV vaccine studies is very limited. In the current study, both immune sera and monoclonal antibodies from vaccinated humans showed not only high-level ADCC and ADCP activities but also cross-subtype ADCC and ADCP activities when a polyvalent DNA prime-protein boost

  3. GABAB and adenosine receptors mediate enhancement of the K+ current, IAHP, by reducing adenylyl cyclase activity in rat CA3 hippocampal neurons.

    Science.gov (United States)

    Gerber, U; Gähwiler, B H

    1994-11-01

    1. Gamma-aminobuturic acid-B (GABAB) and adenosine A1 receptors, which are expressed in hippocampal pyramidal cells, are linked to pertussis toxin-sensitive G-proteins known to be coupled negatively to the enzyme adenylyl cyclase. This study investigates the electrophysiological consequences of adenylyl cyclase inhibition in response to stimulation of these receptors. 2. Single-electrode voltage-clamp recordings were obtained from CA3 pyramidal cells in rat hippocampal slice cultures in presence of tetrodotoxin. The calcium-dependent potassium current (IAHP), which is very sensitive to intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP), was used as an electrophysiological indicator of adenylyl cyclase activity. 3. Application of baclofen (10 microM), a selective agonist at GABAB receptors, or adenosine (50 microM) each resulted in a transient decrease followed by a significant enhancement in the amplitude of evoked IAHP. The initial reduction in amplitude of IAHP probably reflects inadequacies in voltage clamp of electronically distant dendritic sites, due to the shunting caused by concomitant activation of potassium conductance by baclofen/adenosine. Comparable increases in membrane conductance in response to the GABAA agonist, muscimol, caused a similar reduction in IAHP. The enhancement of IAHP is consistent with an inhibition of constitutively active adenylyl cyclase. 4. The receptor mediating the responses to adenosine was identified as belonging to the A1 subtype on the basis of its sensitivity to the selective antagonist 8-cyclopentyl-1,3-dipropylxanthine.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. Contribution of priority PAHs and POPs to Ah receptor-mediated activities in sediment samples from the River Elbe Estuary, Germany.

    Science.gov (United States)

    Otte, Jens C; Keiter, Steffen; Faßbender, Christopher; Higley, Eric B; Rocha, Paula Suares; Brinkmann, Markus; Wahrendorf, Dierk-Steffen; Manz, Werner; Wetzel, Markus A; Braunbeck, Thomas; Giesy, John P; Hecker, Markus; Hollert, Henner

    2013-01-01

    The estuary of the River Elbe between Hamburg and the North Sea (Germany) is a sink for contaminated sediment and suspended particulate matter (SPM). One major concern is the effect of human activities on the hydrodynamics, particularly the intensive dredging activities in this area that may result in remobilization of sediment-bound pollutants. The aim of this study was to identify pollutants contributing to the toxicological risk associated with re-suspension of sediments in the Elbe Estuary by use of an effect-directed analysis that combines chemical and biological analyses in with specific fractionation techniques. Sediments were collected from sites along the Elbe Estuary and a site from a small harbor basin of the Elbe Estuary that is known to be polluted. The sixteen priority EPA-PAHs were quantified in organic extracts of sediments. In addition, dioxin equivalents of sediments were investigated by use of the 7-ethoxyresorufin O-deethylase assay with RTL-W1 cells and the Ah receptor-mediated luciferase transactivation assay with H4IIE-luc cells. Quantification of the 16 priority PAHs revealed that sediments were moderately contaminated at all of the sites in the Elbe River Estuary (Elbe River into its estuary. Successful identification of a significant portion of dioxin-like activity to priority PAHs in complex environmental samples such as sediments has rarely been reported.

  5. Aryl hydrocarbon receptor-mediated and estrogenic activities of oxygenated polycyclic aromatic hydrocarbons and azaarenes originally identified in extracts of river sediments.

    Science.gov (United States)

    Machala, M; Ciganek, M; Bláha, L; Minksová, K; Vondráck, J

    2001-12-01

    Reproductive dysfunction in wildlife populations can be a result of environmental contaminants binding to aryl hydrocarbon receptor (AhR) or estrogenic receptors. Signaling by both types of receptors can be affected by polycyclic aromatic hydrocarbons (PAHs), which are potential endocrine disruptors. However, our knowledge regarding the effects of oxygenated (oxy)-PAHs and azaarenes on AhR-mediated and estrogenic activities is incomplete. In the present study, we have identified 9-fluorenone, anthrone, anthraquinone, benzanthrone, benz[a]anthracene-7,12-dione, benz[c]acridine, and dibenz[a,h]acridine as prevalent oxy-PAHs and azaarenes found in river sediments. Their concentrations in sediment samples ranged from 2.1 to 165.2 ng g(-1) for oxy-PAHs and up to 27.3 ng g(-1) for azaarenes. Their relative AhR-inducing and estrogenic potencies were quantified in vitro using two cell lines that were stably transfected with a luciferase reporter gene system and expressed as induction equivalency factors (IEFs). The only oxy-PAHs with detectable levels of in vitro AhR-mediated activity were benzanthrone and benz[a]anthracene-7,12-dione. However, their IEFs were approximately three to four orders of magnitude lower than those of benzo[a]pyrene. On the other hand, azaarenes showed a strong AhR-mediated activity, with dibenzo[a,h]acridine being a far more potent inducer of activity than benzo[a]pyrene. Benzanthrone, benz[a]anthracene-7,12-dione, anthraquinone, and benz[a]acridine were weak inducers of in vitro estrogenic activity, with IEFs similar to that of benzo[a]pyrene. Based on concentrations and relative potencies, our results suggest that dibenzo[a,h]acridine can significantly contribute to the overall AhR-mediated activity in river sediments, whereas the remaining compounds do not. No studied compound was found to contribute significantly to estrogen receptor-mediated activity in vitro.

  6. Peroxisome Proliferator-Activated Receptor : Mediates the Effects of High-Fat Diet on Hepatic Gene Expression

    NARCIS (Netherlands)

    Patsouris, D.A.; Reddy, J.K.; Müller, M.R.; Kersten, A.H.

    2006-01-01

    Peroxisome proliferator-activated receptors (PPARs) are transcription factors involved in the regulation of numerous metabolic processes. The PPAR isotype is abundant in liver and activated by fasting. However, it is not very clear what other nutritional conditions activate PPAR. To examine whether

  7. ADP stimulates human endothelial cell migration via P2Y1 nucleotide receptor-mediated mitogen-activated protein kinase pathways.

    Science.gov (United States)

    Shen, Jianzhong; DiCorleto, Paul E

    2008-02-29

    Extensive research on the role of ADP in platelet activation led to the design of new anti-thrombotic drugs, such as clopidogrel (Plavix; sanofi-aventis); however, very little is known about the ADP-preferring nucleotide receptors (P2Y1, P2Y12, and P2Y13) in endothelium. Here, we show that ADP stimulates migration of cultured human umbilical vein endothelial cells (HUVECs) in both Boyden chamber and in vitro wound repair assays. This promigratory effect was mimicked by 2-MeSADP, but not by AMP, and was inhibited by MRS2179 (P2Y1 receptor antagonist) but not by AR-C69931MX (P2Y12/13 receptor antagonist). RT-PCR revealed abundant P2Y1, barely detectable P2Y12, and absent P2Y13 receptor message in these cells. In addition, both ADP and 2-MeSADP, but not AMP, activated the mitogen-activated protein kinase pathways as evidenced by increased phosphorylation of extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK), and p38 kinase. ADP also stimulated phosphorylation of p90RSK, a downstream substrate of phosphorylated ERK1/2, and induced phosphorylation of such transcription factors downstream of the JNK and p38 pathways as c-Jun and activating transcription factor-2. These signaling events were inhibited by MRS2179 but not by AR-C69931MX. Furthermore, blockade of the ERK or JNK pathways by U0126 and SP600125, respectively, abolished ADP- and 2-MeSADP-stimulated HUVEC migration. However, inhibition of the p38 pathway by SB203580 partially suppressed ADP- and 2-MeSADP-induced HUVEC migration. We conclude that ADP promotes human endothelial cell migration by activating P2Y1 receptor-mediated MAPK pathways, possibly contributing to reendothelialization and angiogenesis after vascular injury.

  8. Pregnane-X-Receptor Mediates The Anti-inflammatory Activities of Rifaximin on Detoxification Pathways in Intestinal Epithelial cells

    OpenAIRE

    Mencarelli, Andrea; Migliorati, Marco; Barbanti, Miriam; Cipriani, Sabrina; Palladino, Giuseppe; Distrutti, Eleonora; Renga, Barbara; Fiorucci, Stefano

    2010-01-01

    Abstract The pregnane-X-receptor (PXR) is master gene overseeing detoxification of wide number of xenobiotics and is critical for maintenance of intestinal integrity. The intestinal expression of genes involved in cellular detoxification is down-regulated in patients with inflammatory bowel diseases (IBD). Rifaximin, is a non absorbable antibiotic endowed with a PXR agonistic activity. In the present study we have investigated whether rifaximin activates PXR in primary human colon ...

  9. P2X7 receptor-mediated PARP1 activity regulates astroglial death in the rat hippocampus following status epilepticus

    Directory of Open Access Journals (Sweden)

    Ji Yang eKim

    2015-09-01

    Full Text Available Poly(ADP-ribose polymerase-1 (PARP1 plays a regulatory role in apoptosis, necrosis, and other cellular processes after injury. Recently, we revealed that PARP1 regulates the differential neuronal/astroglial responses to pilocarpine-induced status epilepticus (SE in the distinct brain regions. In addition, P2X7 receptor (P2X7R, an ATP-gated ion channel, activation accelerates astroglial apoptosis, while it attenuates clasmatodendrosis (lysosome-derived autophagic astroglial death. Therefore, we investigated whether P2X7R regulates regional specific astroglial PARP1 expression/activation in response to SE. In the present study, P2X7R activation exacerbates SE-induced astroglial apoptosis, while P2X7R inhibition attenuates it accompanied by increasing PARP1 activity in the molecular layer of the dentate gyrus following SE. In the CA1 region, however, P2X7R inhibition deteriorates SE-induced clasmatodendrosis via PARP1 activation following SE. Taken together, our findings suggest that P2X7R function may affect SE-induced astroglial death by regulating PARP1 activation/expression in regional-specific manner. Therefore, the selective modulation of P2X7R-mediated PARP1 functions may be a considerable strategy for controls in various types of cell deaths.

  10. Long-term exposure to PGE2 causes homologous desensitization of receptor-mediated activation of protein kinase A.

    Science.gov (United States)

    Malty, Ramy Habashy; Hudmon, Andy; Fehrenbacher, Jill C; Vasko, Michael R

    2016-07-11

    Acute exposure to prostaglandin E2 (PGE2) activates EP receptors in sensory neurons which triggers the cAMP-dependent protein kinase A (PKA) signaling cascade resulting in enhanced excitability of the neurons. With long-term exposure to PGE2, however, the activation of PKA does not appear to mediate persistent PGE2-induced sensitization. Consequently, we examined whether homologous desensitization of PGE2-mediated PKA activation occurs after long-term exposure of isolated sensory neurons to the eicosanoid. Sensory neuronal cultures were harvested from the dorsal root ganglia of adult male Sprague-Dawley rats. The cultures were pretreated with vehicle or PGE2 and used to examine signaling mechanisms mediating acute versus persistent sensitization by exposure to the eicosanoid using enhanced capsaicin-evoked release of immunoreactive calcitonin gene-related peptide (iCGRP) as an endpoint. Neuronal cultures chronically exposed to vehicle or PGE2 also were used to study the ability of the eicosanoid and other agonists to activate PKA and whether long-term exposure to the prostanoid alters expression of EP receptor subtypes. Acute exposure to 1 μM PGE2 augments the capsaicin-evoked release of iCGRP, and this effect is blocked by the PKA inhibitor H-89. After 5 days of exposure to 1 μM PGE2, administration of the eicosanoid still augments evoked release of iCGRP, but the effect is not attenuated by inhibition of PKA or by inhibition of PI3 kinases. The sensitizing actions of PGE2 after acute and long-term exposure were attenuated by EP2, EP3, and EP4 receptor antagonists, but not by an EP1 antagonist. Exposing neuronal cultures to 1 μM PGE2 for 12 h to 5 days blocks the ability of PGE2 to activate PKA. The offset of the desensitization occurs within 24 h of removal of PGE2 from the cultures. Long-term exposure to PGE2 also results in desensitization of the ability of a selective EP4 receptor agonist, L902688 to activate PKA, but does not alter the ability of

  11. Participation of the cell polarity protein PALS1 to T-cell receptor-mediated NF-κB activation.

    Science.gov (United States)

    Carvalho, Gabrielle; Poalas, Konstantinos; Demian, Catherine; Hatchi, Emeline; Vazquez, Aimé; Bidère, Nicolas

    2011-03-30

    Beside their established function in shaping cell architecture, some cell polarity proteins were proposed to participate to lymphocyte migration, homing, scanning, as well as activation following antigen receptor stimulation. Although PALS1 is a central component of the cell polarity network, its expression and function in lymphocytes remains unknown. Here we investigated whether PALS1 is present in T cells and whether it contributes to T Cell-Receptor (TCR)-mediated activation. By combining RT-PCR and immunoblot assays, we found that PALS1 is constitutively expressed in human T lymphocytes as well as in Jurkat T cells. siRNA-based knockdown of PALS1 hampered TCR-induced activation and optimal proliferation of lymphocyte. We further provide evidence that PALS1 depletion selectively hindered TCR-driven activation of the transcription factor NF-κB. The cell polarity protein PALS1 is expressed in T lymphocytes and participates to the optimal activation of NF-κB following TCR stimulation.

  12. Participation of the cell polarity protein PALS1 to T-cell receptor-mediated NF-κB activation.

    Directory of Open Access Journals (Sweden)

    Gabrielle Carvalho

    Full Text Available BACKGROUND: Beside their established function in shaping cell architecture, some cell polarity proteins were proposed to participate to lymphocyte migration, homing, scanning, as well as activation following antigen receptor stimulation. Although PALS1 is a central component of the cell polarity network, its expression and function in lymphocytes remains unknown. Here we investigated whether PALS1 is present in T cells and whether it contributes to T Cell-Receptor (TCR-mediated activation. METHODOLOGY/PRINCIPAL FINDINGS: By combining RT-PCR and immunoblot assays, we found that PALS1 is constitutively expressed in human T lymphocytes as well as in Jurkat T cells. siRNA-based knockdown of PALS1 hampered TCR-induced activation and optimal proliferation of lymphocyte. We further provide evidence that PALS1 depletion selectively hindered TCR-driven activation of the transcription factor NF-κB. CONCLUSIONS: The cell polarity protein PALS1 is expressed in T lymphocytes and participates to the optimal activation of NF-κB following TCR stimulation.

  13. Grape seed extract regulates androgen receptor-mediated transcription in prostate cancer cells through potent anti-histone acetyltransferase activity.

    Science.gov (United States)

    Park, Si Yong; Lee, Yoo-Hyun; Choi, Kyung-Chul; Seong, Ah-Reum; Choi, Hyo-Kyoung; Lee, Ok-Hee; Hwang, Han-Joon; Yoon, Ho-Geun

    2011-01-01

    Histone acetylation, which is regulated by histone acetyltransferases (HATs) and deacetylases, is an epigenetic mechanism that influences eukaryotic transcription. Significant changes in histone acetylation are associated with cancer; therefore, manipulating the acetylation status of key gene targets is likely crucial for effective cancer therapy. Grape seed extract (GSE) has a known protective effect against prostate cancer. Here, we showed that GSE significantly inhibited HAT activity by 30-80% in vitro (P cancer cells by measuring luciferase activity using a pGL3-PSA construct bearing the AR element in the human prostate cancer cell line LNCaP (P cancer cell growth, and implicate GSE as a novel candidate for therapeutic activity against prostate cancer.

  14. Histone H4 Lys 20 methyltransferase SET8 promotes androgen receptor-mediated transcription activation in prostate cancer

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Lushuai [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Li, Yanyan; Du, Fengxia [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); Han, Xiao [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Li, Xiaohua [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); Niu, Yuanjie [Chawnshang Chang Sex Hormone Research Center, Tianjin Institute of Urology, Tianjin Medical University, Tianjin 300070 (China); Ren, Shancheng, E-mail: renshancheng@gmail.com [Department of Urology, Shanghai Changhai Hospital, Second Military Medical University, Shanghai 200433 (China); Sun, Yingli, E-mail: sunyl@big.ac.cn [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China)

    2014-07-18

    Highlights: • Dihydrotestosterone stimulates H4K20me1 enrichment at the PSA promoter. • SET8 promotes AR-mediated transcription activation. • SET8 interacts with AR and promotes cell proliferation. - Abstract: Histone methylation status in different lysine residues has an important role in transcription regulation. The effect of H4K20 monomethylation (H4K20me1) on androgen receptor (AR)-mediated gene transcription remains unclear. Here we show that AR agonist stimulates the enrichment of H4K20me1 and SET8 at the promoter of AR target gene PSA in an AR dependent manner. Furthermore, SET8 is crucial for the transcription activation of PSA. Co-immunoprecipitation analyses demonstrate that SET8 interacts with AR. Therefore, we conclude that SET8 is involved in AR-mediated transcription activation, possibly through its interaction with AR and H4K20me1 modification.

  15. The CB1 receptor mediates the peripheral effects of ghrelin on AMPK activity but not on growth hormone release.

    Science.gov (United States)

    Kola, Blerina; Wittman, Gábor; Bodnár, Ibolya; Amin, Faisal; Lim, Chung Thong; Oláh, Márk; Christ-Crain, Mirjam; Lolli, Francesca; van Thuijl, Hinke; Leontiou, Chrysanthia A; Füzesi, Tamás; Dalino, Paolo; Isidori, Andrea M; Harvey-White, Judith; Kunos, George; Nagy, György M; Grossman, Ashley B; Fekete, Csaba; Korbonits, Márta

    2013-12-01

    This study aimed to investigate whether the growth hormone release and metabolic effects of ghrelin on AMPK activity of peripheral tissues are mediated by cannabinoid receptor type 1 (CB1) and the central nervous system. CB1-knockout (KO) and/or wild-type mice were injected peripherally or intracerebroventricularly with ghrelin and CB1 antagonist rimonabant to study tissue AMPK activity and gene expression (transcription factors SREBP1c, transmembrane protein FAS, enzyme PEPCK, and protein HSL). Growth hormone levels were studied both in vivo and in vitro. Peripherally administered ghrelin in liver, heart, and adipose tissue AMPK activity cannot be observed in CB1-KO or CB1 antagonist-treated mice. Intracerebroventricular ghrelin treatment can influence peripheral AMPK activity. This effect is abolished in CB1-KO mice and by intracerebroventricular rimonabant treatment, suggesting that central CB1 receptors also participate in the signaling pathway that mediates the effects of ghrelin on peripheral tissues. Interestingly, in vivo or in vitro growth hormone release is intact in response to ghrelin in CB1-KO animals. Our data suggest that the metabolic effects of ghrelin on AMPK in peripheral tissues are abolished by the lack of functional CB1 receptor via direct peripheral effect and partially through the central nervous system, thus supporting the existence of a possible ghrelin-cannabinoid-CB1-AMPK pathway.

  16. Xeno-oestrogens Bisphenol A and Diethylstilbestrol Selectively Activating Androgen Receptor Mediated AREs-TATA Reporter System

    Institute of Scientific and Technical Information of China (English)

    WU Jing; WEI Wei; YANG Nan-yang; SHEN Xiao-yan; TSUJI Ichiro; YAMAMURA Takaki; LI Jiang

    2013-01-01

    We cloned the three androgen response elements(AREs,including AREⅠ,AREⅡ,and AREⅢ) with a core transactivation TATA element of the prostate-specific antigen(PSA) promoter into pGL2 basic vector to create an artificial pGL2/AREs-TATA reporter system,which was applied to evaluating the effects of different xenooestrogens[bisphenol A(BPA),4-nonylphenol(4-NP),dichlorodiphenyl trichloroethane(DDT) or diethylstilbestrol (DES)] on androgen receptor(AR) abnormal activation to regulate PSA expression and cell proliferation.In all the three AREs,AREⅢ-TATA displayed as a major element responsive to AR-mediated DHT stimulation of PSA promoter.Therefore,pGL2/AREⅢ-TATA reporter was adopted to analyze the activation capacity of AR activated by four different xeno-oestrogens.The activation ofpGL2/AREⅢ-TATA reporter by each xeno-oestrogen was analyzed in two different cell lines,one was HEK293T(Human Embryonic Kidney 293T) cell line,and the other was AR stably expressed DU145 cell line,which was produced by infecting AR with pLenti-puro-AR into the prostate cancer DU145 cells and that were scanned with puromycin and tested by AR antibody.In both the two cell lines,BPA or DES significantly induced AR-mediated transcriptional activity of AREⅢ-TATA reporter,whereas DDT or 4-nonylphenol did not.Moreover,AR-mediated cell proliferation in response to each of four xeno-oestrogens was measured in MTT assays in both HEK293T cell or AR stably expressed DU145 cell lines.BPA or DES,as an AR inducer,exhibited an enhanced effect in cell proliferation,rather than the effect of DDT or 4-NP,in both cell lines.Finally,we demonstrated that BPA or DES stimulated PSA expression and enhanced the recruitment of AR onto thePSA promoter,resulting in stronger binding to AREⅢ sites.Taken together,four xeno-oestrogens were identified to have different activities on AR.BPA and DES are demonstrated to be androgenic effectors in the regulation of PSA activation or cell proliferation.

  17. Activation of epidermal growth factor receptor mediates mucin production stimulated by p40, a Lactobacillus rhamnosus GG-derived protein.

    Science.gov (United States)

    Wang, Lihong; Cao, Hailong; Liu, Liping; Wang, Bangmao; Walker, W Allan; Acra, Sari A; Yan, Fang

    2014-07-18

    The mucus layer coating the gastrointestinal tract serves as the first line of intestinal defense against infection and injury. Probiotics promote mucin production by goblet cells in the intestine. p40, a Lactobacillus rhamnosus GG-derived soluble protein, has been shown to transactivate the EGF receptor (EGFR) in intestinal epithelial cells, which is required for inhibition of apoptosis and preservation of barrier function in the colon, thereby ameliorating intestinal injury and colitis. Because activation of EGFR has been shown to up-regulate mucin production in goblet cells, the purpose of this study was to investigate the effects and mechanisms of p40 regulation of mucin production. p40 activated EGFR and its downstream target, Akt, in a concentration-dependent manner in LS174T cells. p40 stimulated Muc2 gene expression and mucin production in LS174T cells, which were abolished by inhibition of EGFR kinase activity, down-regulation of EGFR expression by EGFR siRNA transfection, or suppression of Akt activation. Treatment with p40 increased mucin production in the colonic epithelium, thus thickening the mucus layer in the colon of wild type, but not of Egfr(wa5) mice, which have a dominant negative mutation in the EGFR kinase domain. Furthermore, inhibition of mucin-type O-linked glycosylation suppressed the effect of p40 on increasing mucin production and protecting intestinal epithelial cells from TNF-induced apoptosis in colon organ culture. Thus, these results suggest that p40-stimulated activation of EGFR mediates up-regulation of mucin production, which may contribute to the mechanisms by which p40 protects the intestinal epithelium from injury.

  18. Presynaptic facilitatory adenosine A2A receptors mediate fade induced by neuromuscular relaxants that exhibit anticholinesterase activity.

    Science.gov (United States)

    Bornia, Elaine Cs; Correia-de-Sá, Paulo; Alves-Do-Prado, Wilson

    2011-03-01

    1. Pancuronium, cisatracurium and vecuronium are antinicotinic agents that, in contrast with d-tubocurarine and hexamethonium, exhibit anticholinesterase activity. Pancuronium-, cisatracurium- and vecuronium-induced fade results from blockade of facilitatory nicotinic receptors on motor nerves, but fade produced by such agents also depends on the presynaptic activation of inhibitory muscarinic M2 receptors by acetylcholine released from motor nerve terminals and activation of inhibitory adenosine A1 receptors by adenosine released from motor nerves and muscles. The participation of presynaptic facilitatory A2A receptors in fade caused by pancuronium, cisatracurium and vecuronium has not yet been investigated. In the present study, we determined the effects of ZM241385, an antagonist of presynaptic facilitatory A2A receptors, on fade produced by these neuromuscular relaxants in the rat phrenic nerve-diaphragm (PND) preparation. 2. The muscles were stimulated indirectly at 75±3Hz to induce a sustained tetanizing muscular contraction. The lowest concentration at which each antinicotinic agent produced fade without modifying initial tetanic tension (presynaptic action) was determined. 3. d-Tubocurarine-induced fade occurred only at 55 nmol/L, a concentration that also reduced maximal tetanic tension (post-synaptic action). At 10 nmol/L, ZM 241385 alone did not produce fade, but it did attenuate pancuronium (0.32 μmol/L)-, cisatracurium (0.32 μmol/L)- and vecuronium (0.36 μmol/L)-induced fade. 4. The fade induced by the 'pure' antinicotinic agents d-tubocurarine (55 nmol/L) and hexamethonium (413 μmol/L) was not altered by 10 nmol/L ZM 241385, indicating that presynaptic adenosine A2A receptors play a significant role in the fade produced by antinicotinic agents when such agents have anticholinesterase activity.

  19. Activation of retinoid receptor-mediated signaling ameliorates diabetes-induced cardiac dysfunction in Zucker diabetic rats.

    Science.gov (United States)

    Guleria, Rakeshwar S; Singh, Amar B; Nizamutdinova, Irina T; Souslova, Tatiana; Mohammad, Amin A; Kendall, Jonathan A; Baker, Kenneth M; Pan, Jing

    2013-04-01

    Diabetic cardiomyopathy (DCM) is a significant contributor to the morbidity and mortality associated with diabetes and metabolic syndrome. Retinoids, through activation of retinoic acid receptor (RAR) and retinoid x receptor (RXR), have been linked to control glucose and lipid homeostasis, with effects on obesity and diabetes. However, the functional role of RAR and RXR in the development of DCM remains unclear. Zucker diabetic fatty (ZDF) and lean rats were treated with Am580 (RARα agonist) or LGD1069 (RXR agonist) for 16 weeks, and cardiac function and metabolic alterations were determined. Hyperglycemia, hyperlipidemia and insulin resistance were observed in ZDF rats. Diabetic cardiomyopathy was characterized in ZDF rats by increased oxidative stress, apoptosis, fibrosis, inflammation, activation of MAP kinases and NF-κB signaling and diminished Akt phosphorylation, along with decreased glucose transport and increased cardiac lipid accumulation, and ultimately diastolic dysfunction. Am580 and LGD1069 attenuated diabetes-induced cardiac dysfunction and the pathological alterations, by improving glucose tolerance and insulin resistance; facilitating Akt activation and glucose utilization, and attenuating oxidative stress and interrelated MAP kinase and NF-κB signaling pathways. Am580 inhibited body weight gain, attenuated the increased cardiac fatty acid uptake, β-oxidation and lipid accumulation in the hearts of ZDF rats. However, LGD1069 promoted body weight gain, hyperlipidemia and cardiac lipid accumulation. In conclusion, our data suggest that activation of RAR and RXR may have therapeutic potential in the treatment of diabetic cardiomyopathy. However, further studies are necessary to clarify the role of RAR and RXR in the regulation of lipid metabolism and homeostasis.

  20. Inflammation and N-formyl peptide receptors mediate the angiogenic activity of human vitreous humour in proliferative diabetic retinopathy.

    Science.gov (United States)

    Rezzola, Sara; Corsini, Michela; Chiodelli, Paola; Cancarini, Anna; Nawaz, Imtiaz M; Coltrini, Daniela; Mitola, Stefania; Ronca, Roberto; Belleri, Mirella; Lista, Liliana; Rusciano, Dario; De Rosa, Mario; Pavone, Vincenzo; Semeraro, Francesco; Presta, Marco

    2017-04-01

    Angiogenesis and inflammation characterise proliferative diabetic retinopathy (PDR), a major complication of diabetes mellitus. However, the impact of inflammation on the pathogenesis of PDR neovascularisation has not been elucidated. Here, we assessed the capacity of PDR vitreous fluid to induce pro-angiogenic/proinflammatory responses in endothelium and the contribution of the inflammation-related pattern recognition N-formyl peptide receptors (FPRs) in mediating these responses. Pooled and individual pars plana vitrectomy-derived PDR vitreous fluid ('PDR vitreous') samples were assessed in endothelial cell proliferation, motility, sprouting and morphogenesis assays, and for the capacity to induce proinflammatory transcription factor activation, reactive oxygen species production, intercellular junction disruption and leucocyte-adhesion molecule upregulation in these cells. In vivo, the pro-angiogenic/proinflammatory activity of PDR vitreous was tested in murine Matrigel plug and chick embryo chorioallantoic membrane (CAM) assays. Finally, the FPR inhibitors Boc-Phe-Leu-Phe-Leu-Phe (Boc-FLFLF) and Ac-L-Arg-Aib-L-Arg-L-Cα(Me)Phe-NH2 tetrapeptide (UPARANT) were evaluated for their capacity to affect the biological responses elicited by PDR vitreous. PDR vitreous activates a pro-angiogenic/proinflammatory phenotype in endothelial cells. Accordingly, PDR vitreous triggers a potent angiogenic/inflammatory response in vivo. Notably, the different capacity of individual PDR vitreous samples to induce neovessel formation in the CAM correlates with their ability to recruit infiltrating CD45(+) cells. Finally, the FPR inhibitor Boc-FLFLF and the novel FPR antagonist UPARANT inhibit neovessel formation and inflammatory responses triggered by PDR vitreous in the CAM assay. This study provides evidence that inflammation mediates the angiogenic activity of PDR vitreous and paves the way for the development of FPR-targeting anti-inflammatory/anti-angiogenic approaches for PDR

  1. NOD1 cooperates with TLR2 to enhance T cell receptor-mediated activation in CD8 T cells.

    Directory of Open Access Journals (Sweden)

    Blandine C Mercier

    Full Text Available Pattern recognition receptors (PRR, like Toll-like receptors (TLR and NOD-like receptors (NLR, are involved in the detection of microbial infections and tissue damage by cells of the innate immune system. Recently, we and others have demonstrated that TLR2 can additionally function as a costimulatory receptor on CD8 T cells. Here, we establish that the intracytosolic receptor NOD1 is expressed and functional in CD8 T cells. We show that C12-iEDAP, a synthetic ligand for NOD1, has a direct impact on both murine and human CD8 T cells, increasing proliferation and effector functions of cells activated via their T cell receptor (TCR. This effect is dependent on the adaptor molecule RIP2 and is associated with an increased activation of the NF-κB, JNK and p38 signaling pathways. Furthermore, we demonstrate that NOD1 stimulation can cooperate with TLR2 engagement on CD8 T cells to enhance TCR-mediated activation. Altogether our results indicate that NOD1 might function as an alternative costimulatory receptor in CD8 T cells. Our study provides new insights into the function of NLR in T cells and extends to NOD1 the recent concept that PRR stimulation can directly control T cell functions.

  2. Endothelial nuclear lamina is not required for glucocorticoid receptor nuclear import but does affect receptor-mediated transcription activation.

    Science.gov (United States)

    Nayebosadri, Arman; Ji, Julie Y

    2013-08-01

    The lamina serves to maintain the nuclear structure and stiffness while acting as a scaffold for heterochromatin and many transcriptional proteins. Its role in endothelial mechanotransduction, specifically how nuclear mechanics impact gene regulation under shear stress, is not fully understood. In this study, we successfully silenced lamin A/C in bovine aortic endothelial cells to determine its role in both glucocorticoid receptor (GR) nuclear translocation and glucocorticoid response element (GRE) transcriptional activation in response to dexamethasone and shear stress. Nuclear translocation of GR, an anti-inflammatory nuclear receptor, in response to dexamethasone or shear stress (5, 10, and 25 dyn/cm(2)) was observed via time-lapse cell imaging and quantified using a Bayesian image analysis algorithm. Transcriptional activity of the GRE promoter was assessed using a dual-luciferase reporter plasmid. We found no dependence on nuclear lamina for GR translocation from the cytoplasm into the nucleus. However, the absence of lamin A/C led to significantly increased expression of luciferase under dexamethasone and shear stress induction as well as changes in histone protein function. PCR results for NF-κB inhibitor alpha (NF-κBIA) and dual specificity phosphatase 1 (DUSP1) genes further supported our luciferase data with increased expression in the absence of lamin. Our results suggest that absence of lamin A/C does not hinder passage of GR into the nucleus, but nuclear lamina is important to properly regulate GRE transcription. Nuclear lamina, rather than histone deacetylase (HDAC), is a more significant mediator of shear stress-induced transcriptional activity, while dexamethasone-initiated transcription is more HDAC dependent. Our findings provide more insights into the molecular pathways involved in nuclear mechanotransduction.

  3. Interplay between BCL10, MALT1 and IkappaBalpha during T-cell-receptor-mediated NFkappaB activation.

    Science.gov (United States)

    Carvalho, Gabrielle; Le Guelte, Armelle; Demian, Catherine; Vazquez, Aimé; Gavard, Julie; Bidère, Nicolas

    2010-07-15

    T-cell-receptor (TCR) signalling to NFkappaB requires the assembly of a large multiprotein complex containing the serine/threonine kinase CK1alpha, the scaffold protein CARMA1, the heterodimer BCL10-MALT1 (the CBM complex) and the IkappaB kinase complex (IKK). Although the mechanisms regulating recruitment and activation of IKK within the CBM microenvironment have been extensively studied, there is little understanding of how IKK subsequently binds and phosphorylates IkappaBalpha, the inhibitor of NFkappaB, to promote IkappaBalpha ubiquitylation and proteasomal degradation. Here, we show that BCL10, MALT1 and IKK inducibly associate with IkappaBalpha in a complex that is physically distinct from the early CK1alpha-CBM signalosome. This IkappaBalpha-containing complex probably maturates from the CBM, because siRNA-based knockdown of CARMA1, CK1alpha and BCL10 hampered its assembly, leading to a reduction in NFkappaB activation. By contrast, CK1alpha normally recruited both BCL10 and ubiquitylated species of MALT1 when IkappaBalpha levels were reduced. However, knockdown of IkappaBalpha led to an altered ubiquitylation profile of BCL10-MALT1 combined with a defect in MALT1 reorganisation within large cytoplasmic structures, suggesting that, following stimulation, IkappaBalpha might also participate in MALT1 recycling. Altogether, our data suggest a two-step mechanism to connect active IKK to IkappaBalpha, and further unveil a potential role for IkappaBalpha in resetting TCR-mediated signalling.

  4. Isoflavones made simple - genistein's agonist activity for the beta-type estrogen receptor mediates their health benefits.

    Science.gov (United States)

    McCarty, Mark Frederick

    2006-01-01

    Soy isoflavones, the focus of much research and controversy, are often referred to as "weak estrogens". In fact, genistein is a relatively potent agonist for the recently characterized beta isoform of the estrogen receptor (ERbeta). The low nanomolar serum concentrations of unconjugated free genistein achieved with high-nutritional intakes of soy isoflavones are near the binding affinity of genistein for this receptor, but are about an order of magnitude lower than genistein's affinity for the "classical" alpha isoform of the estrogen receptor (ERalpha). Moreover, these concentrations are far too low to inhibit tyrosine kinases or topoisomerase II, in vitro activities of genistein often cited as potential mediators of its physiological effects. The thesis that these physiological effects are in fact mediated by ERbeta activation provides a satisfying rationale for genistein's clinical activities. Hepatocytes do not express ERbeta; this explains why soy isoflavones, unlike oral estrogen, neither modify serum lipids nor provoke the prothrombotic effects associated with increased risk for thromboembolic disorders. The lack of uterotrophic activity of soy isoflavones reflects the fact that ERalpha is the exclusive mediator of estrogen's impact in this regard. Vascular endothelium expresses both ERalpha and ERbeta, each of which has the potential to induce and activate nitric oxide synthase; this may account for the favorable influence of soy isoflavones on endothelial function in postmenopausal women and ovariectomized rats. The ERbeta expressed in osteoblasts may mediate the reported beneficial impact of soy isoflavones on bone metabolism. Suggestive evidence that soy-rich diets decrease prostate cancer risk, accords well with the observation that ERbeta appears to play an antiproliferative role in healthy prostate. In the breast, ERalpha promotes epithelial proliferation, whereas ERbeta has a restraining influence in this regard - consistent with the emerging view

  5. Activation of the cannabinoid type-1 receptor mediates the anticonvulsant properties of cannabinoids in the hippocampal neuronal culture models of acquired epilepsy and status epilepticus.

    Science.gov (United States)

    Blair, Robert E; Deshpande, Laxmikant S; Sombati, Sompong; Falenski, Katherine W; Martin, Billy R; DeLorenzo, Robert J

    2006-06-01

    Cannabinoids have been shown to have anticonvulsant properties, but no studies have evaluated the effects of cannabinoids in the hippocampal neuronal culture models of acquired epilepsy (AE) and status epilepticus (SE). This study investigated the anticonvulsant properties of the cannabinoid receptor agonist R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolol[1,2,3 de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone (WIN 55,212-2) in primary hippocampal neuronal culture models of both AE and SE. WIN 55,212-2 produced dose-dependent anticonvulsant effects against both spontaneous recurrent epileptiform discharges (SRED) (EC50 = 0.85 microM) and SE (EC50 = 1.51 microM), with total suppression of seizure activity at 3 microM and of SE activity at 5 microM. The anticonvulsant properties of WIN 55,212-2 in these preparations were both stereospecific and blocked by the cannabinoid type-1 (CB1) receptor antagonist N-(piperidin-1-yl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride (SR141716A; 1 microM), showing a CB1 receptor-dependent pathway. The inhibitory effect of WIN 55,212-2 against low Mg2+-induced SE is the first observation in this model of total suppression of SE by a selective pharmacological agent. The clinically used anticonvulsants phenytoin and phenobarbital were not able to abolish low Mg2+-induced SE at concentrations up to 150 microM. The results from this study show CB1 receptor-mediated anticonvulsant effects of the cannabimimetic WIN 55,212-2 against both SRED and low Mg2+-induced SE in primary hippocampal neuronal cultures and show that these in vitro models of AE and SE may represent powerful tools to investigate the molecular mechanisms mediating the effects of cannabinoids on neuronal excitability.

  6. Receptor-mediated activation of gastric vagal afferents by glucagon-like peptide-1 in the rat

    DEFF Research Database (Denmark)

    Bucinskaite, V; Tolessa, T; Pedersen, J

    2009-01-01

    The vagus nerve plays a role in mediating effects of the two glucagon-like peptides GLP-1 and GLP-2 on gastrointestinal growth, functions and eating behaviour. To obtain electrophysiological and molecular evidence for the contribution of afferent pathways in chemoreception from the gastrointestinal...... tract, afferent mass activity in the ventral gastric branch of the vagus nerve and gene expression of GLP-1 receptors and GLP-2 receptors in the nodose ganglion were examined in Sprague-Dawley rats. Intravenous administration of GLP-1 (30-1000 pmol kg(-1)), reaching high physiological plasma...... afferent nerves mediate sensory input from the gastrointestinal tract or pancreas; either directly or indirectly via the release of another mediator. GLP-2 receptors appear not be functionally expressed on vagal afferents....

  7. PSD-95 regulates D1 dopamine receptor resensitization, but not receptor-mediated Gs-protein activation

    Institute of Scientific and Technical Information of China (English)

    Peihua Sun; Jingru Wang; Weihua Gu; Wei Cheng; Guo-zhang Jin; Eitan Friedman; Jie Zheng; Xuechu Zhen

    2009-01-01

    The present study aims to define the role of postsynaptic density (PSD)-95 in the regulation of dopamine (DA) receptor function. We found that PSD-95 physically associates with either D1 or D2 DA receptors in co-transfected HEK-293 cells. Stimulation of DA receptors altered the association between D1 receptor and PSD-95 in a time-depen-dent manner. Functional assays indicated that PSD-95 co-expression did not affect D1 receptor-stimulated cAMP pro-duction, Gs-protein activation or receptor desensitization. However, PSD-95 accelerated the recovery of internalized membrane receptors by promoting receptor recycling, thus resulting in enhanced resensitization of internalized D1 receptors. Our results provide a novel mechanism for regulating DA receptor recycling that may play an important role in postsynaptic DA functional modulation and synaptic neuroplasticity.

  8. CDK11{sup p58} represses vitamin D receptor-mediated transcriptional activation through promoting its ubiquitin-proteasome degradation

    Energy Technology Data Exchange (ETDEWEB)

    Chi, Yayun; Hong, Yi; Zong, Hongliang; Wang, Yanlin; Zou, Weiying; Yang, Junwu; Kong, Xiangfei; Yun, Xiaojing [Gene Research Center, Shanghai Medical College and Institutes of Biomedical, Shanghai 200032 (China); Gu, Jianxin, E-mail: jxgu@shmu.edu.cn [Gene Research Center, Shanghai Medical College and Institutes of Biomedical, Shanghai 200032 (China)

    2009-08-28

    Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily and regulates transcription of target genes. In this study, we identified CDK11{sup p58} as a novel protein involved in the regulation of VDR. CDK11{sup p58}, a member of the large family of p34cdc2-related kinases, is associated with cell cycle progression, tumorigenesis, and apoptotic signaling. Our study demonstrated that CDK11{sup p58} interacted with VDR and repressed VDR-dependent transcriptional activation. Furthermore, overexpression of CDK11{sup p58} decreased the stability of VDR through promoting its ubiquitin-proteasome-mediated degradation. Taken together, these results suggest that CDK11{sup p58} is involved in the negative regulation of VDR.

  9. Pregnane-X-receptor mediates the anti-inflammatory activities of rifaximin on detoxification pathways in intestinal epithelial cells.

    Science.gov (United States)

    Mencarelli, Andrea; Migliorati, Marco; Barbanti, Miriam; Cipriani, Sabrina; Palladino, Giuseppe; Distrutti, Eleonora; Renga, Barbara; Fiorucci, Stefano

    2010-12-01

    The pregnane-X-receptor (PXR) is master gene overseeing detoxification of wide number of xenobiotics and is critical for maintenance of intestinal integrity. The intestinal expression of genes involved in cellular detoxification is down-regulated in patients with inflammatory bowel diseases (IBD). Rifaximin is a non-absorbable antibiotic endowed with a PXR agonistic activity. In the present study we have investigated whether rifaximin activates PXR in primary human colon epithelial cells and human colon biopsies and assessed whether this antibiotic antagonizes the effect of tumor necrosis factor (TNF)-α on expression of PXR and PXR-related genes. Present results demonstrate that primary colon epithelial cells express PXR and that their exposure to rifaximin induces the expression of genes involved in cellular detoxification. Exposure to TNFα reduces the expression of PXR mRNA as well as expression of its target genes. This inhibitory effect was prevented by that co-treatment with rifaximin. Knocking down the expression of PXR in colon epithelial cells by an anti-PXR siRNA, abrogated the counter-regulatory effects exerted by rifaximin on cell exposed to TNFα. Finally, ex vivo exposure of colon biopsies obtained from ulcerative colitis patients to rifaximin increased the expression of genes involved in xenobiotics metabolism. In aggregate, these data illustrate that rifaximin increases the expression of PXR and PXR-regulated genes involved in the metabolism and excretion of xenobiotics and antagonizes the effects of TNFα in intestinal epithelial cells and colon biopsies. These non-antibiotic effects of rifaximin could contribute to the maintenance of the intestinal barrier integrity against xenobiotics and products generated by luminal bacteria.

  10. Apparent receptor-mediated activation of Ca2+-dependent conductive Cl- transport by shark-derived polyaminosterols.

    Science.gov (United States)

    Chernova, Marina N; Vandorpe, David H; Clark, Jeffrey S; Williams, Jon I; Zasloff, Michael A; Jiang, Lianwei; Alper, Seth L

    2005-12-01

    The shark liver antimicrobial polyaminosterol squalamine is an angiogenesis inhibitor under clinical investigation as an anti-cancer agent and as a treatment for the choroidal neovascularization associated with macular degeneration of the retina. The related polyaminosterol MSI-1436 is an appetite suppressant that decreases systemic insulin resistance. However, the mechanisms of action of these polyaminosterols are unknown. We report effects of MSI-1436 on Xenopus oocytes consistent with the existence of a receptor for polyaminosterols. MSI-1436 activates bidirectional, trans-chloride-independent Cl- flux in Xenopus oocytes. At least part of this DIDS-sensitive Cl- flux is conductive, as measured using two-electrode voltage-clamp and on-cell patch-clamp techniques. MSI-1436 also elevates cytosolic Ca2+ concentration ([Ca2+]) and increases bidirectional 45Ca2+ flux. Activation of Cl- flux and elevation of cytosolic [Ca2+] by MSI-1436 both are accelerated by lowering bath Ca2+ and are not acutely inhibited by extracellular EGTA. Elevation of cytosolic [Ca2+] by MSI-1436 requires heparin-sensitive intracellular Ca2+ stores. Although injected EGTA abolishes the increased conductive Cl- flux, that Cl- flux is not dependent on heparin-sensitive stores. In low-bath Ca2+ conditions, several structurally related polyaminosterols act as strong agonists or weak agonists of conductive Cl- flux in oocytes. Weak agonist polyaminosterols antagonize the strong agonist, MSI-1436, but upon addition of the conductive Cl- transport inhibitor DIDS, they are converted into strong agonists. Together, these properties operationally define a polyaminosterol receptor at or near the surface of the Xenopus oocyte, provide an initial description of receptor signaling, and suggest routes toward further understanding of a novel class of appetite suppressants and angiogenesis inhibitors.

  11. P2X7 Receptor Mediates Spinal Microglia Activation of Visceral Hyperalgesia in a Rat Model of Chronic Pancreatitis.

    Science.gov (United States)

    Liu, Pei-Yi; Lee, I-Hui; Tan, Ping-Heng; Wang, Yen-Po; Tsai, Chia-Fen; Lin, Han-Chieh; Lee, Fa-Yauh; Lu, Ching-Liang

    2015-11-01

    Molecular mechanisms underlying the activated spinal microglia in association with the pain in chronic pancreatitis (CP) remain unknown. We tested whether P2X7R on spinal microglia mediates the pathogenesis of visceral pain using a CP rat model. The CP model was induced via intraductal injection of 2% trinitrobenzene sulfonic acid into male Sprague-Dawley rats. Hyperalgesia was assessed based on the mechanical sensitivity to Von-Frey filaments (VFFs), and nocifensive behaviors were measured in response to electrical stimulation of the pancreas. Three weeks after CP induction, spinal cord samples were harvested for immunostaining, immunoblot, and real-time polymerase chain reaction analyses of the P2X7R. Changes in nocifensive behaviors and associated molecular effectors were assessed by blocking spinal cord P2X7R pharmacologically using the selective P2X7R antagonist brilliant blue G (BBG) or genetically using short interfering RNA (siRNA). CP induced a significant up-regulation of spinal P2X7R expression, which colocalized with a microglial marker (OX-42). Intrathecal administration of BBG significantly attenuated CP-related visceral hyperalgesia in response to VFF-mediated or electrical stimulation of the pancreas, which was associated with suppressed spinal expression of P2X7R and inhibited activation of spinal microglia. Intrathecal injection of siRNA to knock down P2X7R expression in the spinal cord would suppress the nociceptive behaviors in CP rats. Spinal microglia P2X7R mediates central sensitization of chronic visceral pain in CP. BBG may represent an effective drug for the treatment of chronic pain in CP patients.

  12. Estrogen-, androgen- and aryl hydrocarbon receptor mediated activities in passive and composite samples from municipal waste and surface waters.

    Science.gov (United States)

    Jálová, V; Jarošová, B; Bláha, L; Giesy, J P; Ocelka, T; Grabic, R; Jurčíková, J; Vrana, B; Hilscherová, K

    2013-09-01

    Passive and composite sampling in combination with in vitro bioassays and identification and quantification of individual chemicals were applied to characterize pollution by compounds with several specific modes of action in urban area in the basin of two rivers, with 400,000 inhabitants and a variety of industrial activities. Two types of passive samplers, semipermeable membrane devices (SPMD) for hydrophobic contaminants and polar organic chemical integrative samplers (POCIS) for polar compounds such as pesticides and pharmaceuticals, were used to sample wastewater treatment plant (WWTP) influent and effluent as well as rivers upstream and downstream of the urban complex and the WWTP. Compounds with endocrine disruptive potency were detected in river water and WWTP influent and effluent. Year-round, monthly assessment of waste waters by bioassays documented estrogenic, androgenic and dioxin-like potency as well as cytotoxicity in influent waters of the WWTP and allowed characterization of seasonal variability of these biological potentials in waste waters. The WWTP effectively removed cytotoxic compounds, xenoestrogens and xenoandrogens. There was significant variability in treatment efficiency of dioxin-like potency. The study indicates that the WWTP, despite its up-to-date technology, can contribute endocrine disrupting compounds to the river. Riverine samples exhibited dioxin-like, antiestrogenic and antiandrogenic potencies. The study design enabled characterization of effects of the urban complex and the WWTP on the river. Concentrations of PAHs and contaminants and specific biological potencies sampled by POCIS decreased as a function of distance from the city. © 2013.

  13. Structure of unliganded HSV gD reveals a mechanism for receptor-mediated activation of virus entry

    Energy Technology Data Exchange (ETDEWEB)

    Krummenacher, Claude; Supekar, Vinit M.; Whitbeck, J. Charles; Lazear, Eric; Connolly, Sarah A.; Eisenberg, Roselyn J.; Cohen, Gary H.; Wiley, Don C.; Carfi, Andrea (UPENN); (IRBM); (CHLMM)

    2010-07-19

    Herpes simplex virus (HSV) entry into cells requires binding of the envelope glycoprotein D (gD) to one of several cell surface receptors. The 50 C-terminal residues of the gD ectodomain are essential for virus entry, but not for receptor binding. We have determined the structure of an unliganded gD molecule that includes these C-terminal residues. The structure reveals that the C-terminus is anchored near the N-terminal region and masks receptor-binding sites. Locking the C-terminus in the position observed in the crystals by an intramolecular disulfide bond abolished receptor binding and virus entry, demonstrating that this region of gD moves upon receptor binding. Similarly, a point mutant that would destabilize the C-terminus structure was nonfunctional for entry, despite increased affinity for receptors. We propose that a controlled displacement of the gD C-terminus upon receptor binding is an essential feature of HSV entry, ensuring the timely activation of membrane fusion.

  14. Contribution of priority PAHs and POPs to Ah receptor-mediated activities in sediment samples from the River Elbe Estuary, Germany.

    Directory of Open Access Journals (Sweden)

    Jens C Otte

    Full Text Available The estuary of the River Elbe between Hamburg and the North Sea (Germany is a sink for contaminated sediment and suspended particulate matter (SPM. One major concern is the effect of human activities on the hydrodynamics, particularly the intensive dredging activities in this area that may result in remobilization of sediment-bound pollutants. The aim of this study was to identify pollutants contributing to the toxicological risk associated with re-suspension of sediments in the Elbe Estuary by use of an effect-directed analysis that combines chemical and biological analyses in with specific fractionation techniques. Sediments were collected from sites along the Elbe Estuary and a site from a small harbor basin of the Elbe Estuary that is known to be polluted. The sixteen priority EPA-PAHs were quantified in organic extracts of sediments. In addition, dioxin equivalents of sediments were investigated by use of the 7-ethoxyresorufin O-deethylase assay with RTL-W1 cells and the Ah receptor-mediated luciferase transactivation assay with H4IIE-luc cells. Quantification of the 16 priority PAHs revealed that sediments were moderately contaminated at all of the sites in the Elbe River Estuary (<0.02-0.906 µg/g dw. Sediments contained relatively small concentrations of dioxin equivalents (Bio-TEQ with concentrations ranging from 15.5 to 322 pg/g dw, which were significantly correlated with dioxin equivalents calculated based on toxicity reference values and concentrations of PAH. The concentration of Bio-TEQ at the reference site exceeded 200,000 pg/g dw. In a potency balance the 16 PAHs explained between 47 and 118% of the Bio-TEQ in the luciferase assay, which can be explained by the constant input of PAHs bound to SPM from the upper course of the Elbe River into its estuary. Successful identification of a significant portion of dioxin-like activity to priority PAHs in complex environmental samples such as sediments has rarely been reported.

  15. Contribution of Priority PAHs and POPs to Ah Receptor-Mediated Activities in Sediment Samples from the River Elbe Estuary, Germany

    Science.gov (United States)

    Otte, Jens C.; Keiter, Steffen; Faßbender, Christopher; Higley, Eric B.; Rocha, Paula Suares; Brinkmann, Markus; Wahrendorf, Dierk-Steffen; Manz, Werner; Wetzel, Markus A.; Braunbeck, Thomas; Giesy, John P.; Hecker, Markus; Hollert, Henner

    2013-01-01

    The estuary of the River Elbe between Hamburg and the North Sea (Germany) is a sink for contaminated sediment and suspended particulate matter (SPM). One major concern is the effect of human activities on the hydrodynamics, particularly the intensive dredging activities in this area that may result in remobilization of sediment-bound pollutants. The aim of this study was to identify pollutants contributing to the toxicological risk associated with re-suspension of sediments in the Elbe Estuary by use of an effect-directed analysis that combines chemical and biological analyses in with specific fractionation techniques. Sediments were collected from sites along the Elbe Estuary and a site from a small harbor basin of the Elbe Estuary that is known to be polluted. The sixteen priority EPA-PAHs were quantified in organic extracts of sediments. In addition, dioxin equivalents of sediments were investigated by use of the 7-ethoxyresorufin O-deethylase assay with RTL-W1 cells and the Ah receptor-mediated luciferase transactivation assay with H4IIE-luc cells. Quantification of the 16 priority PAHs revealed that sediments were moderately contaminated at all of the sites in the Elbe River Estuary (<0.02–0.906 µg/g dw). Sediments contained relatively small concentrations of dioxin equivalents (Bio-TEQ) with concentrations ranging from 15.5 to 322 pg/g dw, which were significantly correlated with dioxin equivalents calculated based on toxicity reference values and concentrations of PAH. The concentration of Bio-TEQ at the reference site exceeded 200,000 pg/g dw. In a potency balance the 16 PAHs explained between 47 and 118% of the Bio-TEQ in the luciferase assay, which can be explained by the constant input of PAHs bound to SPM from the upper course of the Elbe River into its estuary. Successful identification of a significant portion of dioxin-like activity to priority PAHs in complex environmental samples such as sediments has rarely been reported. PMID:24146763

  16. Receptor-Mediated Drug Delivery Systems Targeting to Glioma

    Directory of Open Access Journals (Sweden)

    Shanshan Wang

    2015-12-01

    Full Text Available Glioma has been considered to be the most frequent primary tumor within the central nervous system (CNS. The complexity of glioma, especially the existence of the blood-brain barrier (BBB, makes the survival and prognosis of glioma remain poor even after a standard treatment based on surgery, radiotherapy, and chemotherapy. This provides a rationale for the development of some novel therapeutic strategies. Among them, receptor-mediated drug delivery is a specific pattern taking advantage of differential expression of receptors between tumors and normal tissues. The strategy can actively transport drugs, such as small molecular drugs, gene medicines, and therapeutic proteins to glioma while minimizing adverse reactions. This review will summarize recent progress on receptor-mediated drug delivery systems targeting to glioma, and conclude the challenges and prospects of receptor-mediated glioma-targeted therapy for future applications.

  17. Genomic variation in the MMP-1 promoter influences estrogen receptor mediated activity in a mechanically activated environment: potential implications for microgravity risk assessment

    Science.gov (United States)

    Thaler, John; Myers, Ken; Lu, Ting; Hart, David

    examine the potential impact of the 1G/2G SNP on the cellular response to mechanical loading. HIG-82 cells are estrogen receptor (ER) negative and were transiently transfected with SV40 expression vectors for either ER-α or ER-β isoforms. Cells grown on glass slides were also co-transfected with either a 1G or 2G MMP-1 promoter-luciferase construct. Transfected cells were subjected to dynamic shear stress in a Flexcell Streamer Shear Stress Device. The dynamic loading regime was 0.5 Hz, 10 dyn/cm2 shear for 1 minute followed by 14 minutes rest and repeated for 8 hrs. A Promega Dual Luciferase Reporter Assay System was used to assess MMP-1 promoter activity. Results: Shear stress loading increased both 1G and 2G MMP-1 promoter activity compared to unloaded controls, however the 2G promoter had significantly higher rates of expression than the 1G promoter across all loading regimes and ER co-transfections. Transfection with ER-β resulted in higher MMP-1 promoter activity than that in cells expressing ER-α or in ER-neg cells. Conclusions: Specific genomic variations can lead to differences in cellular responses to changes in mechanical loading environments such as are encountered in microgravity environments or earth-based analogs. These genomic differences may predispose individuals to greater risk of bone loss. It is important to understand the combined effects of mechanical loading, genetic variation and sex hormones on bone maintenance so that risks can be identified for microgravity or analog environments, and specific interventions developed to counteract such risk or even exclude some individuals from prolonged space environments due to the extent of the risk.

  18. Neuroprotection of GluR5-containing kainate receptor activation against ischemic brain injury through decreasing tyrosine phosphorylation of N-methyl-D-aspartate receptors mediated by Src kinase.

    Science.gov (United States)

    Xu, Jie; Liu, Yong; Zhang, Guang-Yi

    2008-10-24

    Previous studies indicate that cerebral ischemia breaks the dynamic balance between excitatory and inhibitory inputs. The neural excitotoxicity induced by ionotropic glutamate receptors gain the upper hand during ischemia-reperfusion. In this paper, we investigate whether GluR5 (glutamate receptor 5)-containing kainate receptor activation could lead to a neuroprotective effect against ischemic brain injury and the related mechanism. The results showed that (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA), a selective GluR5 agonist, could suppress Src tyrosine phosphorylation and interactions among N-methyl-D-aspartate (NMDA) receptor subunit 2A (NR2A), postsynaptic density protein 95 (PSD-95), and Src and then decrease NMDA receptor activation through attenuating tyrosine phosphorylation of NR2A and NR2B. More importantly, ATPA had a neuroprotective effect against ischemia-reperfusion-induced neuronal cell death in vivo. However, four separate drugs were found to abolish the effects of ATPA. These were selective GluR5 antagonist NS3763; GluR5 antisense oligodeoxynucleotides; CdCl(2), a broad spectrum blocker of voltage-gated calcium channels; and bicuculline, an antagonist of gamma-aminobutyric acid A (GABA(A)) receptor. GABA(A) receptor agonist muscimol could attenuate Src activation and interactions among NR2A, PSD-95 and Src, resulting the suppression of NMDA receptor tyrosine phosphorylation. Moreover, patch clamp recording proved that the activated GABA(A) receptor could inhibit NMDA receptor-mediated whole-cell currents. Taken together, the results suggest that during ischemia-reperfusion, activated GluR5 may facilitate Ca(2+)-dependent GABA release from interneurons. The released GABA can activate postsynaptic GABA(A) receptors, which then attenuates NMDA receptor tyrosine phosphorylation through inhibiting Src activation and disassembling the signaling module NR2A-PSD-95-Src. The final result of this process is that the pyramidal

  19. Receptor-Mediated Signalling in Aspergillus fumigatus

    Directory of Open Access Journals (Sweden)

    C. M Grice

    2013-02-01

    Full Text Available Aspergillus fumigatus is the most pathogenic species among the Aspergilli, and the major fungal agent of human pulmonary infection. To prosper in diverse ecological niches, Aspergilli have evolved numerous mechanisms for adaptive gene regulation, some of which are also crucial for mammalian infection. Among the molecules which govern such responses, integral membrane receptors are thought to be the most amenable to therapeutic modulation. This is due to the localisation of these molecular sensors at the periphery of the fungal cell, and to the prevalence of small molecules and licensed drugs which target receptor-mediated signalling in higher eukaryotic cells. In this review we highlight the progress made in characterising receptor-mediated environmental adaptation in A. fumigatus and its relevance for pathogenicity in mammals. By presenting a first genomic survey of integral membrane proteins in this organism, we highlight an abundance of putative 7TMD receptors, the majority of which remain uncharacterised. Given the dependency of A. fumigatus upon stress adaptation for colonisation and infection of mammalian hosts, and the merits of targeting receptor-mediated signalling as an antifungal strategy, a closer scrutiny of sensory perception and signal transduction in this organism is warranted.

  20. Activation of EphA receptors mediates the recruitment of the adaptor protein Slap, contributing to the downregulation of N-methyl-D-aspartate receptors.

    Science.gov (United States)

    Semerdjieva, Sophia; Abdul-Razak, Hayder H; Salim, Sharifah S; Yáñez-Muñoz, Rafael J; Chen, Philip E; Tarabykin, Victor; Alifragis, Pavlos

    2013-04-01

    Regulation of the activity of N-methyl-d-aspartate receptors (NMDARs) at glutamatergic synapses is essential for certain forms of synaptic plasticity underlying learning and memory and is also associated with neurotoxicity and neurodegenerative diseases. In this report, we investigate the role of Src-like adaptor protein (Slap) in NMDA receptor signaling. We present data showing that in dissociated neuronal cultures, activation of ephrin (Eph) receptors by chimeric preclustered eph-Fc ligands leads to recruitment of Slap and NMDA receptors at the sites of Eph receptor activation. Interestingly, our data suggest that prolonged activation of EphA receptors is as efficient in recruiting Slap and NMDA receptors as prolonged activation of EphB receptors. Using established heterologous systems, we examined whether Slap is an integral part of NMDA receptor signaling. Our results showed that Slap does not alter baseline activity of NMDA receptors and does not affect Src-dependent potentiation of NMDA receptor currents in Xenopus oocytes. We also demonstrate that Slap reduces excitotoxic cell death triggered by activation of NMDARs in HEK293 cells. Finally, we present evidence showing reduced levels of NMDA receptors in the presence of Slap occurring in an activity-dependent manner, suggesting that Slap is part of a mechanism that homeostatically modulates the levels of NMDA receptors.

  1. Interleukin-10 and prostaglandin E2 have complementary but distinct suppressive effects on Toll-like receptor-mediated dendritic cell activation in ovarian carcinoma.

    Science.gov (United States)

    Brencicova, Eva; Jagger, Ann L; Evans, Hayley G; Georgouli, Mirella; Laios, Alex; Attard Montalto, Steve; Mehra, Gautam; Spencer, Jo; Ahmed, Ahmed A; Raju-Kankipati, Shanti; Taams, Leonie S; Diebold, Sandra S

    2017-01-01

    Dendritic cells (DC) have the potential to instigate a tumour-specific immune response, but their ability to prime naïve lymphocytes depends on their activation status. Thus, for tumour immunotherapy to be effective, the provision of appropriate DC activation stimuli such as Toll-like receptor (TLR) agonists is crucial in order to overcome immunosuppression associated with the tumour microenvironment. To address this, we investigated how ovarian carcinoma (OC)-associated ascites impedes activation of DC by TLR agonists. Our results show that ascites reduces the TLR-mediated up-regulation of CD86 and partially inhibits the production of the pro-inflammatory cytokines interleukin 6 (IL-6), IL-12 and tumour necrosis factor α (TNFα) in monocyte-derived DC from healthy controls. We further observe an impaired T cell stimulatory capacity of DC upon activation with TLR agonists in the presence of ascites, indicating that their functionality is affected by the immunosuppressive factors. We identify IL-10 and prostaglandin E2 (PGE2) as the pivotal immunosuppressive components in OC-associated ascites compromising TLR-mediated DC activation. Interestingly, IL-10 is present in both ascites from patients with malignant OC and in peritoneal fluid from patients with benign ovarian conditions and both fluids have similar ability to reduce TLR-mediated DC activation. However, depletion of IL-10 from ascites revealed that the presence of paracrine IL-10 is not crucial for ascites-mediated suppression of DC activation in response to TLR activation. Unlike IL-10, PGE2 is absent from peritoneal fluid of patients with benign conditions and selectively reduces TNFα induction in response to TLR-mediated activation in the presence of OC-associated ascites. Our study highlights PGE2 as an immunosuppressive component of the malignant OC microenvironment rendering PGE2 a potentially important target for immunotherapy in OC.

  2. Dopamine D2 receptor-mediated Akt/PKB signalling: initiation by the D2S receptor and role in quinpirole-induced behavioural activation.

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    Chen, Han-Ting; Ruan, Nan-Yu; Chen, Jin-Chung; Lin, Tzu-Yung

    2012-09-24

    The short and long isoforms of the dopamine D2 receptor (D2S and D2L respectively) are highly expressed in the striatum. Functional D2 receptors activate an intracellular signalling pathway that includes a cAMP-independent route involving Akt/GSK3 (glycogen synthase kinase 3). To investigate the Akt/GSK3 response to the seldom-studied D2S receptor, we established a rat D2S receptor-expressing cell line [HEK (human embryonic kidney)-293/rD2S]. We found that in HEK-293/rD2S cells, the D2/D3 agonists bromocriptine and quinpirole significantly induced Akt and GSK3 phosphorylation, as well as ERK1/2 (extracellular-signal-regulated kinase 1/2) activation. The D2S receptor-induced Akt signals were profoundly inhibited by the internalization blockers monodansyl cadaverine and concanavalin A. Activation of the D2S receptor in HEK-293/rD2S cells appeared to trigger Akt/phospho-Akt translocation to the cell membrane. In addition to our cell culture experiments, we studied D2 receptor-dependent Akt in vivo by systemic administration of the D2/D3 agonist quinpirole. The results show that quinpirole evoked Akt-Ser473 phosphorylation in the ventral striatum. Furthermore, intra-accumbens administration of wortmannin, a PI3K (phosphoinositide 3-kinase) inhibitor, significantly suppressed the quinpirole-evoked behavioural activation. Overall, we demonstrate that activation of the dopamine D2S receptor stimulates Akt/GSK3 signalling. In addition, in vivo Akt activity in the ventral striatum appears to play an important role in systemic D2/D3 agonist-induced behavioural activation.

  3. Dopamine D2 receptor-mediated Akt/PKB signalling: initiation by the D2S receptor and role in quinpirole-induced behavioural activation

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    Jin‑Chung Chen

    2012-09-01

    Full Text Available The short and long isoforms of the dopamine D2 receptor (D2S and D2L respectively are highly expressed in the striatum. Functional D2 receptors activate an intracellular signalling pathway that includes a cAMP-independent route involving Akt/GSK3 (glycogen synthase kinase 3. To investigate the Akt/GSK3 response to the seldom-studied D2S receptor, we established a rat D2S receptor-expressing cell line [HEK (human embryonic kidney-293/rD2S]. We found that in HEK-293/rD2S cells, the D2/D3 agonists bromocriptine and quinpirole significantly induced Akt and GSK3 phosphorylation, as well as ERK1/2 (extracellular-signal-regulated kinase 1/2 activation. The D2S receptor-induced Akt signals were profoundly inhibited by the internalization blockers monodansyl cadaverine and concanavalin A. Activation of the D2S receptor in HEK-293/rD2S cells appeared to trigger Akt/phospho-Akt translocation to the cell membrane. In addition to our cell culture experiments, we studied D2 receptor-dependent Akt in vivo by systemic administration of the D2/D3 agonist quinpirole. The results show that quinpirole evoked Akt-Ser473 phosphorylation in the ventral striatum. Furthermore, intra-accumbens administration of wortmannin, a PI3K (phosphoinositide 3-kinase inhibitor, significantly suppressed the quinpirole-evoked behavioural activation. Overall, we demonstrate that activation of the dopamine D2S receptor stimulates Akt/GSK3 signalling. In addition, in vivo Akt activity in the ventral striatum appears to play an important role in systemic D2/D3 agonist-induced behavioural activation.

  4. High molecular weight FGF2 isoforms demonstrate canonical receptor-mediated activity and support human embryonic stem cell self-renewal

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    Denis Kole

    2017-05-01

    Full Text Available Basic fibroblast growth factor (FGF2 is a highly pleiotropic member of a large family of growth factors with a broad range of activities, including mitogenesis and angiogenesis (Ornitz et al., 1996; Zhang et al., 2006, and it is known to be essential for maintenance of balance between survival, proliferation, and self-renewal in human pluripotent stem cells (Eiselleova et al., 2009; Zoumaro-Djayoon et al., 2011. A single FGF2 transcript can be translated into five FGF2 protein isoforms, an 18 kDa low molecular weight (LMW isoform and four larger high molecular weight (HMW isoforms (Arese et al., 1999; Arnaud et al., 1999. As they are not generally secreted, high molecular weight (HMW FGF2 isoforms have predominantly been investigated intracellularly; only a very limited number of studies have investigated their activity as extracellular factors. Here we report over-expression, isolation, and biological activity of all recombinant human FGF2 isoforms. We show that HMW FGF2 isoforms can support self-renewal of human embryonic stem cells (hESCs in vitro. Exogenous supplementation with HMW FGF2 isoforms also activates the canonical FGFR/MAPK pathway and induces mitogenic activity in a manner similar to that of the 18 kDa FGF2 isoform. Though all HMW isoforms, when supplemented exogenously, are able to recapitulate LMW FGF2 activity to some degree, it appears that certain isoforms tend to do so more poorly, demonstrating a lesser functional response by several measures. A better understanding of isoform-specific FGF2 effects will lead to a better understanding of developmental and pathological FGF2 signaling.

  5. Assessment of the aryl hydrocarbon receptor-mediated activities of polycyclic aromatic hydrocarbons in a human cell-based reporter gene assay.

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    Vondráček, Jan; Pěnčíková, Kateřina; Neča, Jiří; Ciganek, Miroslav; Grycová, Aneta; Dvořák, Zdeněk; Machala, Miroslav

    2017-01-01

    Activation of the aryl hydrocarbon receptor (AhR)-mediated activity is one of key events in toxicity of polycyclic aromatic hydrocarbons (PAHs). Although various classes of AhR ligands may differentially activate human and rodent AhR, there is presently a lack of data on the human AhR-inducing relative potencies (REPs) of PAHs. Here, we focused on estimation of the AhR-mediated activities of a large set of environmental PAHs in human gene reporter AZ-AhR cell line, with an aim to develop the human AhR-based REP values with potential implications for risk assessment of PAHs. The previously identified weakly active PAHs mostly failed to activate the AhR in human cells. The order for REPs of individual PAHs in human cells largely corresponded with the available data from rodent-based experimental systems; nevertheless, we identified differences up to one order of magnitude in REP values of PAHs between human and rodent cells. Higher REP values were found in human cells for some important environmental contaminants or suspected carcinogens, such as indeno[1,2,3-cd]pyrene, benz[a]anthracene or benzo[b]fluoranthene, while lower REP values were determined for methyl-substituted PAHs. Our results also indicate that a different rate of metabolism for individual PAHs in human vs. rodent cells may affect estimation of REP values in human cell-based assay, and potentially alter toxicity of some compounds, such as benzofluoranthenes, in humans. We applied the AZ-AhR assay to evaluation of the AhR-mediated activity of complex mixtures of organic compounds associated with diesel exhaust particles, and we identified the polar compounds present in these mixtures as being particularly highly active in human cells, as compared with rodent cells. The present data suggest that differences may exist between the AhR-mediated potencies of PAHs in human and rodent cells, and that the AhR-mediated effects of polar PAH derivatives and metabolites in human cell models deserve further

  6. Alcohol Activates TGF-Beta but Inhibits BMP Receptor-Mediated Smad Signaling and Smad4 Binding to Hepcidin Promoter in the Liver

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    Lisa Nicole Gerjevic

    2012-01-01

    Full Text Available Hepcidin, a key regulator of iron metabolism, is activated by bone morphogenetic proteins (BMPs. Mice pair-fed with regular and ethanol-containing L. De Carli diets were employed to study the effect of alcohol on BMP signaling and hepcidin transcription in the liver. Alcohol induced steatosis and TGF-beta expression. Liver BMP2, but not BMP4 or BMP6, expression was significantly elevated. Despite increased BMP expression, the BMP receptor, and transcription factors, Smad1 and Smad5, were not activated. In contrast, alcohol stimulated Smad2 phosphorylation. However, Smad4 DNA-binding activity and the binding of Smad4 to hepcidin promoter were attenuated. In summary, alcohol stimulates TGF-beta and BMP2 expression, and Smad2 phosphorylation but inhibits BMP receptor, and Smad1 and Smad5 activation. Smad signaling pathway in the liver may therefore be involved in the regulation of hepcidin transcription and iron metabolism by alcohol. These findings may help to further understand the mechanisms of alcohol and iron-induced liver injury.

  7. Cholera Toxin Discriminates Between T Helper 1 and 2 Cells in T Cell Receptor-Mediated Activation : Role of cAMP in T Cell Proliferation

    NARCIS (Netherlands)

    Muñoz, Eduardo; Zubiaga, Ana M.; Merrow, Martha; Sauter, Nicholas P.; Huber, Brigitte T.

    1990-01-01

    CD4+ T helper (Th) clones can be divided into interleukin 2 (IL2)-secreting Th1 and IL-4-secreting Th2 cells. We show in the present report that these two Th subsets have different activation requirements for lymphokine production and proliferation: namely, cholera toxin (CT) as well as forskolin in

  8. Cholera Toxin Discriminates Between T Helper 1 and 2 Cells in T Cell Receptor-Mediated Activation : Role of cAMP in T Cell Proliferation

    NARCIS (Netherlands)

    Muñoz, Eduardo; Zubiaga, Ana M.; Merrow, Martha; Sauter, Nicholas P.; Huber, Brigitte T.

    1990-01-01

    CD4+ T helper (Th) clones can be divided into interleukin 2 (IL2)-secreting Th1 and IL-4-secreting Th2 cells. We show in the present report that these two Th subsets have different activation requirements for lymphokine production and proliferation: namely, cholera toxin (CT) as well as forskolin

  9. Effect-directed analysis of Ah receptor-mediated activities caused by PAHs in suspended particulate matter sampled in flood events.

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    Wölz, J; Brack, W; Moehlenkamp, C; Claus, E; Braunbeck, Th; Hollert, H

    2010-07-15

    Suspended particulate matter (SPM) sampled during a flood event in the year 2004 at the rivers Neckar and Rhine (Southwest Germany) was assessed for aryl hydrocarbon receptor (AhR)-mediated activities using EROD induction in the rainbow trout liver cell line RTL-W1. All EROD inductions were normalized to the positive control TCDD and given as bio-TEQ values. Since all samples indicated elevated AhR-mediated toxicities, an effect-directed analysis (EDA) was applied to identify substances causing the effects. In three primary fractions (F1 to F3) non-polar aliphatics, non-polar aromatic substances and more polar substances were separated. Fraction F2, co-eluting with non-polar polyaromatic substances (PACs) including polycyclic aromatic hydrocarbons (PAHs) gave highest AhR-agonistic effects and, thus, were sub-fractionated into seven secondary fractions (F2-1 to F2-7). Fraction F2-1, co-eluting with PCBs and PCDD/Fs, did not cause AhR-agonist activities. F2-2 to F2-4 containing PACs of less than 16 aromatic C-atoms produced minor activities. Highest inductions were detected with fraction F2-5 to F2-7, containing substances of more than 16 aromatic C-atoms (bio-TEQs up to approximately 4500 pg/g). Concentrations and relative potencies (REPs) of priority EPA-PAHs allowed the calculation of chemical toxicity equivalent concentrations (chem-TEQ values). Based on the chem-TEQs, EPA-PAHs explained between 5 and 58% of crude extract bio-TEQs from both rivers. Whereas fractions F2-1 to F2-4 indicated no biological activities, EPA-PAHs in fraction F2-5 to F2-7 accounted for 2 to 137% of AhR-related activities. Copyright 2010 Elsevier B.V. All rights reserved.

  10. Beta 2 subunit-containing nicotinic receptors mediate acute nicotine-induced activation of calcium/calmodulin-dependent protein kinase II-dependent pathways in vivo.

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    Jackson, K J; Walters, C L; Damaj, M I

    2009-08-01

    Nicotine is the addictive component of tobacco, and successful smoking cessation therapies must address the various processes that contribute to nicotine addiction. Thus, understanding the nicotinic acetylcholine receptor (nAChR) subtypes and subsequent molecular cascades activated after nicotine exposure is of the utmost importance in understanding the progression of nicotine dependence. One possible candidate is the calcium/calmodulin-dependent protein kinase II (CaMKII) pathway. Substrates of this kinase include the vesicle-associated protein synapsin I and the transcription factor cAMP response element-binding protein (CREB). The goal of these studies was to examine these postreceptor mechanisms after acute nicotine treatment in vivo. We first show that administration of nicotine increases CaMKII activity in the ventral tegmental area (VTA), nucleus accumbens (NAc), and amygdala. In beta2 nAChR knockout (KO) mice, nicotine does not induce an increase in kinase activity, phosphorylated (p)Synapsin I, or pCREB. In contrast, alpha7 nAChR KO mice show nicotine-induced increases in CaMKII activity and pCREB, similar to their wild-type littermates. Moreover, we show that when animals are pretreated with the CaMKII inhibitors 4-[(2S)-2-[(5-isoquinolinylsulfonyl) methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl]phenyl isoquinolinesulfonic acid ester (KN-62) and N-[2-[[[3-(4-chlorophenyl)-2 propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulphonamide (KN-93), nicotine-induced increase in the kinase activity and pCREB was attenuated in the VTA and NAc, whereas pretreatment with (2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, phosphate) (KN-92), the inactive analog, did not alter the nicotine-induced increase in pCREB. Taken together, these data suggest that the nicotine-induced increase in CaMKII activity may correlate with the nicotine-induced increase in pSynapsin I and pCREB in the VTA and NAc via beta2

  11. Integrin alpha1beta1 controls reactive oxygen species synthesis by negatively regulating epidermal growth factor receptor-mediated Rac activation.

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    Chen, Xiwu; Abair, Tristin D; Ibanez, Maria R; Su, Yan; Frey, Mark R; Dise, Rebecca S; Polk, D Brent; Singh, Amar B; Harris, Raymond C; Zent, Roy; Pozzi, Ambra

    2007-05-01

    Integrins control many cell functions, including generation of reactive oxygen species (ROS) and regulation of collagen synthesis. Mesangial cells, found in the glomerulus of the kidney, are able to produce large amounts of ROS via the NADPH oxidase. We previously demonstrated that integrin alpha1-null mice develop worse fibrosis than wild-type mice following glomerular injury and this is due, in part, to excessive ROS production by alpha1-null mesangial cells. In the present studies, we describe the mechanism whereby integrin alpha1-null mesangial cells produce excessive ROS. Integrin alpha1-null mesangial cells have constitutively increased basal levels of activated Rac1, which result in its increased translocation to the cell membrane, excessive ROS production, and consequent collagen IV deposition. Basal Rac1 activation is a direct consequence of ligand-independent increased epidermal growth factor receptor (EGFR) phosphorylation in alpha1-null mesangial cells. Thus, our study demonstrates that integrin alpha1beta1-EGFR cross talk is a key step in negatively regulating Rac1 activation, ROS production, and excessive collagen synthesis, which is a hallmark of diseases characterized by irreversible fibrosis.

  12. Aryl hydrocarbon receptor mediated activities in road dust from a metropolitan area, Hanoi-Vietnam: contribution of polycyclic aromatic hydrocarbons (PAHs) and human risk assessment.

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    Tuyen, Le Huu; Tue, Nguyen Minh; Suzuki, Go; Misaki, Kentaro; Viet, Pham Hung; Takahashi, Shin; Tanabe, Shinsuke

    2014-09-01

    Dioxin-Responsive Chemical-Activated LUciferase gene eXpression assay (DR-CALUX) was applied to assess the total toxic activity of the mixture of PAHs and related compounds as well as dioxin-related compounds in road dust from urban areas of Hanoi, Vietnam. Road dust from Hanoi contained significantly higher DR-CALUX activities (3 to 39, mean 20 ng CALUX-TEQ/g dw) than those from a rural site (2 to 13, mean 5 ng CALUX-TEQ/g dw). The total concentrations of 24 major PAHs (Σ24PAHs) in urban road dust (0.1 to 5.5, mean 2.5 μg/g dw) were also 6 times higher than those in rural road dust (0.08 to 1.5, mean 0.4 μg/g dw). Diagnostic ratios of PAHs indicated vehicular engine combustion as the major PAH emission source in both sites. PAHs accounted for 0.8 to 60% (mean 10%) and 2 to 76% (mean 20%) of the measured CALUX-TEQs in road dust for Hanoi the rural site, respectively. Benzo[b]-/benzo[k]fluoranthenes were the major TEQ contributors among PAHs, whereas DRCs contributed hydrocarbon receptor agonists in road dust. Significant PAH concentrations in urban dust indicated high mutagenic and carcinogenic potencies. Estimated results of incremental life time cancer risk (ILCR) indicated that Vietnamese populations, especially those in urban areas such as Hanoi, are potentially exposed to high cancer risk via both dust ingestion and dermal contact. This is the first study on the exposure risk of AhR agonists, including PAHs and DRCs, in urban road dust from a developing country using a combined bio-chemical analytical approach.

  13. Histamine H3 receptor activation counteracts adenosine A2A receptor-mediated enhancement of depolarization-evoked [3H]-GABA release from rat globus pallidus synaptosomes.

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    Morales-Figueroa, Guadalupe-Elide; Márquez-Gómez, Ricardo; González-Pantoja, Raúl; Escamilla-Sánchez, Juan; Arias-Montaño, José-Antonio

    2014-08-20

    High levels of histamine H3 receptors (H3Rs) are found in the globus pallidus (GP), a neuronal nucleus in the basal ganglia involved in the control of motor behavior. By using rat GP isolated nerve terminals (synaptosomes), we studied whether H3R activation modified the previously reported enhancing action of adenosine A2A receptor (A2AR) stimulation on depolarization-evoked [(3)H]-GABA release. At 3 and 10 nM, the A2AR agonist CGS-21680 enhanced [(3)H]-GABA release induced by high K(+) (20 mM) and the effect of 3 nM CGS-21680 was prevented by the A2AR antagonist ZM-241385 (100 nM). The presence of presynaptic H3Rs was confirmed by the specific binding of N-α-[methyl-(3)H]-histamine to membranes from GP synaptosomes (maximum binding, Bmax, 1327 ± 79 fmol/mg protein; dissociation constant, Kd, 0.74 nM), which was inhibited by the H3R ligands immepip, clobenpropit, and A-331440 (inhibition constants, Ki, 0.28, 8.53, and 316 nM, respectively). Perfusion of synaptosomes with the H3R agonist immepip (100 nM) had no effect on K(+)-evoked [(3)H]-GABA release, but inhibited the stimulatory action of A2AR activation. In turn, the effect of immepip was blocked by the H3R antagonist clobenpropit, which had no significant effect of its own on K(+)-induced [(3)H]-GABA release. These data indicate that H3R activation selectively counteracts the facilitatory action of A2AR stimulation on GABA release from striato-pallidal projections.

  14. Chylomicron components activate duodenal vagal afferents via a cholecystokinin A receptor-mediated pathway to inhibit gastric motor function in the rat.

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    Glatzle, Jörg; Wang, Yuhua; Adelson, David W; Kalogeris, Theodore J; Zittel, Tilman T; Tso, Patrick; Wei, Jen-Yu; Raybould, Helen E

    2003-07-15

    Nutrients in the intestine initiate changes in secretory and motor function of the gastrointestinal (GI) tract. The nature of the 'sensors' in the intestinal wall is not well characterized. Intestinal lipid stimulates the release of cholecystokinin (CCK) from mucosal entero-endocrine cells, and it is proposed that CCK activates CCK A receptors on vagal afferent nerve terminals. There is evidence that chylomicron components are involved in this lipid transduction pathway. The aim of the present study was to determine (1) the pathway mediating reflex inhibition of gastric motility and (2) activation of duodenal vagal afferents in response to chylomicrons. Mesenteric lymph was obtained from awake rats fitted with lymph fistulas during intestinal perfusion of lipid (Intralipid, 170 micromol h(-1), chylous lymph) or a dextrose and/or electrolyte solution (control lymph). Inhibition of gastric motility was measured manometrically in urethane-anaesthetized recipient rats in response to intra-arterial injection of lymph close to the upper GI tract. Chylous lymph was significantly more potent than control lymph in inhibiting gastric motility. Functional vagal deafferentation by perineural capsaicin or CCK A receptor antagonist (devazepide, 1 mg kg(-1), i.v.) significantly reduced chylous lymph-induced inhibition of gastric motility. The discharge of duodenal vagal afferent fibres was recorded from the dorsal abdominal vagus nerve in an in vitro preparation of the duodenum. Duodenal vagal afferent nerve fibre discharge was significantly increased by close-arterial injection of CCK (1-100 pmol) in 43 of 83 units tested. The discharge of 88% of CCK-responsive fibres was increased by close-arterial injection of chylous lymph; devazepide (100 microg, i.a.) abolished the afferent response to chylous lymph in 83% of these units. These data suggest that in the intestinal mucosa, chylomicrons or their products release endogenous CCK which activates CCK A receptors on vagal afferent

  15. P2Y2 nucleotide receptor-mediated extracellular signal-regulated kinases and protein kinase C activation induces the invasion of highly metastatic breast cancer cells.

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    Eun, So Young; Ko, Young Shin; Park, Sang Won; Chang, Ki Churl; Kim, Hye Jung

    2015-07-01

    Tumor metastasis is considered the main cause of mortality in cancer patients, thus it is important to investigate the differences between high- and low-metastatic cancer cells. Our previous study showed that the highly metastatic breast cancer cell line MDA-MB-231 released higher levels of ATP and exhibited higher P2Y2R activity compared with the low-metastatic breast cancer cell line MCF-7. In addition, P2Y2R activation by ATP released from MDA-MB-231 cells induced hypoxia-inducible factor-1α expression, lysyl oxidase secretion and collagen crosslinking, generating a receptive microenvironment for pre-metastatic niche formation. Thus, in the present study, we investigated which P2Y2R-related signaling pathways are involved in the invasion of breast cancer cells. The highly metastatic breast cancer cells MDA-MB-231 and SK-BR-3 showed higher invasion than MCF-7 and T47D cells at a basal level, which was abolished through P2Y2R knockdown or in the presence of apyrase, an enzyme that hydrolyzes extracellular nucleotides. MDA-MB-231 cells also showed high levels of mesenchymal markers, such as Snail, Vimentin and N-cadherin, but not the epithelial marker E-cadherin and this expression was inhibited through ATP degradation or P2Y2R knockdown. Moreover, SK-BR-3 and MDA-MB231 cells exhibited higher ERK and PKC phosphorylation levels than T47D and MCF-7 cells and upregulated phospho-ERK and -PKC levels in MDA-MB-231 cells were significantly downregulated by apyrase or P2Y2R knockdown. Specific inhibitors of ERK, PKC and PLC markedly reduced the invasion and levels of mesenchymal marker expression in MDA-MB-231 cells. These results suggest that over-activated ERK and PKC pathways are involved in the P2Y2R-mediated invasion of breast cancer cells.

  16. P2Y12 receptor-mediated activation of spinal microglia and p38MAPK pathway contribute to cancer-induced bone pain

    Science.gov (United States)

    Liu, Mingjuan; Yao, Ming; Wang, Hanqi; Xu, Longsheng; Zheng, Ying; Huang, Bing; Ni, Huadong; Xu, Shijie; Zhou, Xuyan; Lian, Qingquan

    2017-01-01

    Background Cancer-induced bone pain (CIBP) is one of the most challenging clinical problems due to a lack of understanding the mechanisms. Recent evidence has demonstrated that activation of microglial G-protein-coupled P2Y12 receptor (P2Y12R) and proinflammatory cytokine production play an important role in neuropathic pain generation and maintenance. However, whether P2Y12R is involved in CIBP remains unknown. Methods The purpose of this study was to investigate the role of P2Y12R in CIBP and its molecular mechanisms. Using the bone cancer model inoculated with Walker 256 tumor cells into the left tibia of Sprague Dawley rat, we blocked spinal P2Y12R through intrathecal administration of its selective antagonist MRS2395 (400 pmol/µL, 15 µL). Results We found that not only the ionized calcium-binding adapter molecule 1 (Iba-1)-positive microglia in the ipsilateral spinal cord but also mechanical allodynia was significantly inhibited. Furthermore, it decreased the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) and the production of proinflammatory cytokines interleukin-1β (IL-1β) and interleukin-6 (IL-6), whereas it increased tumor necrosis factor-α (TNF-α). Conclusion Taken together, our present results suggest that microglial P2Y12R in the spinal cord may contribute to CIBP by the activation of spinal microglia and p38MAPK pathway, thus identifying a potential therapeutic target for the treatment of CIBP.

  17. The Next Generation Non-competitive Active Polyester Nanosystems for Transferrin Receptor-mediated Peroral Transport Utilizing Gambogic Acid as a Ligand.

    Science.gov (United States)

    Saini, P; Ganugula, R; Arora, M; Kumar, M N V Ravi

    2016-01-01

    The current methods for targeted drug delivery utilize ligands that must out-compete endogenous ligands in order to bind to the active site facilitating the transport. To address this limitation, we present a non-competitive active transport strategy to overcome intestinal barriers in the form of tunable nanosystems (NS) for transferrin receptor (TfR) utilizing gambogic acid (GA), a xanthanoid, as its ligand. The NS made using GA conjugated poly(lactide-co-glycolide) (PLGA) have shown non-competitive affinity to TfR evaluated in cell/cell-free systems. The fluorescent PLGA-GA NS exhibited significant intestinal transport and altered distribution profile compared to PLGA NS in vivo. The PLGA-GA NS loaded with cyclosporine A (CsA), a model peptide, upon peroral dosing to rodents led to maximum plasma concentration of CsA at 6 h as opposed to 24 h with PLGA-NS with at least 2-fold higher levels in brain at 72 h. The proposed approach offers new prospects for peroral drug delivery and beyond.

  18. Nonreceptor protein tyrosine and lipid phosphatases in type I fc(epsilon) receptor-mediated activation of mast cells and basophils.

    Science.gov (United States)

    Heneberg, Petr; Dráber, Petr

    2002-08-01

    Protein tyrosine and lipid phosphorylations are early and critical events in type 1 Fc(epsilon) receptor (Fc(epsilon)RI)-mediated activation of mast cells and basophils. Tyrosine phosphorylation of Fc(epsilon)RI subunits as well as other signal transduction molecules reflects the balance between the action of protein tyrosine kinases and phosphatases. Similarly, the phosphate content of inositol phospholipids, involved in the recruitment of signalling molecules to the plasma membrane and the generation of secondary messengers, is the net result of the opposing effects of phosphoinositide kinases and lipid phosphatases. This review summarizes the current understanding of the structural and functional aspects of nonreceptor protein tyrosine phosphatases (SHP-1, SHP-2, HePTP, PTP20, PRL1, PRL2, PTP-MEG1 and PTP-MEG2) and lipid phosphatases (SHIP and SHIP2) in the activation of mast cells and basophils after Fc(epsilon)RI aggregation. New approaches towards a deeper understanding of the role of phosphatases in mast cell physiology are also discussed.

  19. Berberine induces apoptosis in human HSC-3 oral cancer cells via simultaneous activation of the death receptor-mediated and mitochondrial pathway.

    Science.gov (United States)

    Lin, Chin-Chung; Yang, Jai-Sing; Chen, Jin-Tang; Fan, Shang; Yu, Fu-Shun; Yang, Jiun-Long; Lu, Chi-Cheng; Kao, Ming-Ching; Huang, An-Cheng; Lu, Hsu-Feng; Chung, Jing-Gung

    2007-01-01

    Evidence has accumulated that berberine is able to induce cell cycle arrest and apoptosis in many human cancer cell lines. However, there is no available information on the effects of berberine on human oral squamous cell carcinoma. In this study, the effects of berberine on cell growth, apoptosis and cell cycle regulation in human oral squamous carcinoma HSC-3 cells were examined. Berberine induced dose- and time-dependent irreversible inhibition of cell growth and cellular DNA synthesis. This was also confirmed by phase-contrast microscopy which showed that berberine induced morphological changes in HSC-3 cells. Propidium iodide/annexin V staining for flow cytometric analysis showed that berberine-induced apoptosis correlated with caspase-3 activation. Flow cytometric studies of the cell cycle distribution showed that berberine induced mainly G0/G1-phase arrest. Flow cytometric examinations also showed that berberine induced reactive oxygen species (ROS) and Ca2+ production, as well as the dysfunction of mitochondrial membrane potential (MMP), which were correlated with apoptosis. In conclusion, our data support that berberine initially induces an endoplasmic reticulum stress response based on ROS and Ca2+ production which is followed by dysfunctions of the mitochondria, resulting in apoptosis of these oral cancer HSC-3 cells. Prolonged exposure of the HSC-3 cells to berberine causes increased apoptosis through reduced levels of MMP, release of cytochrome c and activation of caspase-3.

  20. Hypocretin/Orexin Peptides Excite Rat Neuroendocrine Dopamine Neurons through Orexin 2 Receptor-Mediated Activation of a Mixed Cation Current

    Science.gov (United States)

    Lyons, David J.; Hellysaz, Arash; Ammari, Rachida; Broberger, Christian

    2017-01-01

    Hypocretin/Orexin (H/O) neurons of the lateral hypothalamus are compelling modulator candidates for the chronobiology of neuroendocrine output and, as a consequence, hormone release from the anterior pituitary. Here we investigate the effects of H/O peptides upon tuberoinfundibular dopamine (TIDA) neurons – cells which control, via inhibition, the pituitary secretion of prolactin. In whole cell recordings performed in male rat hypothalamic slices, application of H/O-A, as well as H/O-B, excited oscillating TIDA neurons, inducing a reversible depolarising switch from phasic to tonic discharge. The H/O-induced inward current underpinning this effect was post-synaptic (as it endured in the presence of tetrodotoxin), appeared to be carried by a Na+-dependent transient receptor potential-like channel (as it was blocked by 2-APB and was diminished by removal of extracellular Na+), and was a consequence of OX2R receptor activation (as it was blocked by the OX2R receptor antagonist TCS OX2 29, but not the OX1R receptor antagonist SB 334867). Application of the hormone, melatonin, failed to alter TIDA membrane potential or oscillatory activity. This first description of the electrophysiological effects of H/Os upon the TIDA network identifies cellular mechanisms that may contribute to the circadian rhythmicity of prolactin secretion. PMID:28145492

  1. Prostanoid EP1 receptors mediate up-regulation of the orphan nuclear receptor Nurr1 by cAMP-independent activation of protein kinase A, CREB and NF-κB

    Science.gov (United States)

    Ji, R; Sanchez, CM; Chou, CL; Chen, XB; Woodward, DF; Regan, JW

    2012-01-01

    BACKGROUND AND PURPOSE Prostaglandin E2 (PGE2) stimulation of the G protein-coupled prostanoid EP1 receptor was found to up-regulate the expression of Nur-related factor 1 (Nurr1) (NR4A2), a transcription factor in the NR4A subfamily of nuclear receptors. The present studies characterize the molecular mechanism of this up-regulation. EXPERIMENTAL APPROACH The expression of Nurr1 was examined by immunoblot analysis, the polymerase chain reaction and reporter gene assays in human embryonic kidney (HEK) cells stably expressing the recombinant EP1 receptor and in SH-SY5Y neuroblastoma cells expressing endogenous EP1 receptors. Signalling pathway inhibitors were used to examine the roles of Rho, PKA, the cAMP response element binding protein (CREB) and NF-κB on the PGE2 stimulated up-regulation of Nurr1. CREB and NF-κB signalling were also examined by immunoblot analysis and reporter gene assays. KEY RESULTS The EP1 receptor mediated up-regulation of Nurr1 was blocked with inhibitors of Rho, PKA, NF-κB and CREB; but PGE2 failed to significantly stimulate intracellular cAMP formation. PGE2 stimulation of the EP1 receptor induced the phosphorylation and activation of CREB and NF-κB, which could be blocked by inhibition of PKA. CONCLUSIONS AND IMPLICATIONS PGE2 stimulation of the human EP1 receptor up-regulates the expression of Nurr1 by a mechanism involving the sequential activation of the Rho, PKA, CREB and NF-κB signalling pathways. EP1 receptors are implicated in tumorigenesis and the up-regulation of Nurr1 may underlie the anti-apoptotic effects of PGE2. PMID:22188298

  2. P2Y12 receptor-mediated activation of spinal microglia and p38MAPK pathway contribute to cancer-induced bone pain

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    Liu MJ

    2017-02-01

    Full Text Available Mingjuan Liu,1 Ming Yao,1,2 Hanqi Wang,1 Longsheng Xu,1 Ying Zheng,1 Bing Huang,1 Huadong Ni,1 Shijie Xu,1 Xuyan Zhou,1 Qingquan Lian2 1Department of Anesthesiology and Pain Medicine, The First Hospital of Jiaxing, The First Affiliated Hospital of Jiaxing University, Jiaxing, 2Department of Anesthesiology, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, People’s Republic of China Background: Cancer-induced bone pain (CIBP is one of the most challenging clinical problems due to a lack of understanding the mechanisms. Recent evidence has demonstrated that activation of microglial G-protein-coupled P2Y12 receptor (P2Y12R and proinflammatory cytokine production play an important role in neuropathic pain generation and maintenance. However, whether P2Y12R is involved in CIBP remains unknown.Methods: The purpose of this study was to investigate the role of P2Y12R in CIBP and its molecular mechanisms. Using the bone cancer model inoculated with Walker 256 tumor cells into the left tibia of Sprague Dawley rat, we blocked spinal P2Y12R through intrathecal administration of its selective antagonist MRS2395 (400 pmol/µL, 15 µL.Results: We found that not only the ionized calcium-binding adapter molecule 1 (Iba-1-positive microglia in the ipsilateral spinal cord but also mechanical allodynia was significantly inhibited. Furthermore, it decreased the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK and the production of proinflammatory cytokines interleukin-1β (IL-1β and interleukin-6 (IL-6, whereas it increased tumor necrosis factor-α (TNF-α.Conclusion: Taken together, our present results suggest that microglial P2Y12R in the spinal cord may contribute to CIBP by the activation of spinal microglia and p38MAPK pathway, thus identifying a potential therapeutic target for the treatment of CIBP. Keywords: P2Y12 receptor, cancer-induced bone pain, p38MAPK pathway, cytokines

  3. Bovine prolactin elevates hTF expression directed by a tissue-specific goat β-casein promoter through prolactin receptor-mediated STAT5a activation.

    Science.gov (United States)

    Jiang, Shizhong; Ren, Zhaorui; Xie, Fei; Yan, Jingbin; Huang, Shuzhen; Zeng, Yitao

    2012-11-01

    Prolactin promotes the expression of exogenous human transferrin gene in the milk of transgenic mice. To elucidate this, a recombinant plasmid of bovine prolactin plus human transferrin vector was co-transfected into cultured murine mammary gland epithelial cells. Prolactin-receptor antagonist and shRNA corresponding to prolactin-receptor mRNA were added into the cell culture mixture to investigate the relations between prolactin-receptor and human transferrin expression after bovine prolactin inducement. Levels of human transferrin in the supernatants were increased under the presentation of bovine prolactin (from 1,076 ± 115 to 1,886 ± 114 pg/ml). With the treatment of prolactin-receptor antagonist or shRNA, human transferrin in cells was declined (1,886 ± 113 vs. 1,233 ± 85 pg/ml or 1,114 ± 75 pg/ml, respectively). An inverse correlation was found between the dosage of prolactin-receptor antagonist and expression level of human transferrin. Real-time qRT-PCR analysis showed that the relative level of signal transducer and activator of transcription 5a (STAT5a) transcript in transfected cells correlated with expression levels of human transferrin in the supernatant of the same cells. Bovine prolactin thus improved the expression of human transferrin through such a possible mechanism that bovine prolactin activated STAT5a transcription expression via combined with prolactin-receptor and suggest a potential utility of the bovine prolactin for efficient expression of valuable pharmaceutical proteins in mammary glands of transgenic animals.

  4. Norepinephrine-Induced Adrenergic Activation Strikingly Increased the Atrial Fibrillation Duration through β1- and α1-Adrenergic Receptor-Mediated Signaling in Mice.

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    Kenji Suita

    Full Text Available Atrial fibrillation (AF is the most common arrhythmias among old people. It causes serious long-term health problems affecting the quality of life. It has been suggested that the autonomic nervous system is involved in the onset and maintenance of AF in human. However, investigation of its pathogenesis and potential treatment has been hampered by the lack of suitable AF models in experimental animals.Our aim was to establish a long-lasting AF model in mice. We also investigated the role of adrenergic receptor (AR subtypes, which may be involved in the onset and duration of AF.Trans-esophageal atrial burst pacing in mice could induce AF, as previously shown, but with only a short duration (29.0 ± 8.1 sec. We found that adrenergic activation by intraperitoneal norepinephrine (NE injection strikingly increased the AF duration. It increased the duration to more than 10 minutes, i.e., by more than 20-fold (656.2 ± 104.8 sec; P<0.001. In this model, a prior injection of a specific β1-AR blocker metoprolol and an α1-AR blocker prazosin both significantly attenuated NE-induced elongation of AF. To further explore the mechanisms underlying these receptors' effects on AF, we assessed the SR Ca(2+ leak, a major trigger of AF, and consequent spontaneous SR Ca(2+ release (SCR in atrial myocytes. Consistent with the results of our in-vivo experiments, both metoprolol and prazosin significantly inhibited the NE-induced SR Ca(2+ leak and SCR. These findings suggest that both β1-AR and α1-AR may play important roles in the development of AF.We have established a long-lasting AF model in mice induced by adrenergic activation, which will be valuable in future AF study using experimental animals, such as transgenic mice. We also revealed the important role of β1- and α1-AR-mediated signaling in the development of AF through in-vivo and in-vitro experiments.

  5. Histamine H3 receptor activation prevents dopamine D1 receptor-mediated inhibition of dopamine release in the rat striatum: a microdialysis study.

    Science.gov (United States)

    Alfaro-Rodriguez, Alfonso; Alonso-Spilsbury, María; Arch-Tirado, Emilio; Gonzalez-Pina, Rigoberto; Arias-Montaño, José-Antonio; Bueno-Nava, Antonio

    2013-09-27

    Histamine H3 receptors (H3Rs) co-localize with dopamine (DA) D1 receptors (D1Rs) on striatal medium spiny neurons and functionally antagonize D1R-mediated responses. The intra-striatal administration of D1R agonists reduces DA release whereas D1R antagonists have the opposite effect. In this work, a microdialysis method was used to study the effect of co-activating D1 and H3 receptors on the release of DA from the rat dorsal striatum. Infusion of the D1R agonist SKF-38393 (0.5 and 1 μM) significantly reduced DA release (26-58%), and this effect was prevented by co-administration of the H3R agonist immepip (10 μM). In turn, the effect of immepip was blocked by the H3R antagonist thioperamide (10 μM). Our results indicate that co-stimulation of post-synaptic D1 and H3 receptors may indirectly regulate basal DA release in the rat striatum and provide in vivo evidence for a functional interaction between D1 and H3 receptors in the basal ganglia.

  6. Assessment of cellular estrogenic activity based on estrogen receptor-mediated reduction of soluble-form catechol-O-methyltransferase (COMT) expression in an ELISA-based system.

    Science.gov (United States)

    Ho, Philip Wing-Lok; Tse, Zero Ho-Man; Liu, Hui-Fang; Lu, Song; Ho, Jessica Wing-Man; Kung, Michelle Hiu-Wai; Ramsden, David Boyer; Ho, Shu-Leong

    2013-01-01

    Xenoestrogens are either natural or synthetic compounds that mimic the effects of endogenous estrogen. These compounds, such as bisphenol-A (BPA), and phthalates, are commonly found in plastic wares. Exposure to these compounds poses major risk to human health because of the potential to cause endocrine disruption. There is huge demand for a wide range of chemicals to be assessed for such potential for the sake of public health. Classical in vivo assays for endocrine disruption are comprehensive but time-consuming and require sacrifice of experimental animals. Simple preliminary in vitro screening assays can reduce the time and expense involved. We previously demonstrated that catechol-O-methyltransferase (COMT) is transcriptionally regulated by estrogen via estrogen receptor (ER). Therefore, detecting corresponding changes of COMT expression in estrogen-responsive cells may be a useful method to estimate estrogenic effects of various compounds. We developed a novel cell-based ELISA to evaluate cellular response to estrogenicity by reduction of soluble-COMT expression in ER-positive MCF-7 cells exposed to estrogenic compounds. In contrast to various existing methods that only detect bioactivity, this method elucidates direct physiological effect in a living cell in response to a compound. We validated our assay using three well-characterized estrogenic plasticizers - BPA, benzyl butyl phthalate (BBP), and di-n-butyl phthalate (DBP). Cells were exposed to either these plasticizers or 17β-estradiol (E2) in estrogen-depleted medium with or without an ER-antagonist, ICI 182,780, and COMT expression assayed. Exposure to each of these plasticizers (10(-9)-10(-7)M) dose-dependently reduced COMT expression (pvitro assays of similar sensitivity. To satisfy the demand for in vitro assays targeting different cellular components, a cell-based COMT assay provides useful initial screening to supplement the current assessments of xenoestrogens for potential estrogenic activity.

  7. Assessment of cellular estrogenic activity based on estrogen receptor-mediated reduction of soluble-form catechol-O-methyltransferase (COMT expression in an ELISA-based system.

    Directory of Open Access Journals (Sweden)

    Philip Wing-Lok Ho

    cellular components, a cell-based COMT assay provides useful initial screening to supplement the current assessments of xenoestrogens for potential estrogenic activity.

  8. Menthol inhibits 5-HT3 receptor-mediated currents.

    Science.gov (United States)

    Ashoor, Abrar; Nordman, Jacob C; Veltri, Daniel; Yang, Keun-Hang Susan; Shuba, Yaroslav; Al Kury, Lina; Sadek, Bassem; Howarth, Frank C; Shehu, Amarda; Kabbani, Nadine; Oz, Murat

    2013-11-01

    The effects of alcohol monoterpene menthol, a major active ingredient of the peppermint plant, were tested on the function of human 5-hydroxytryptamine type 3 (5-HT3) receptors expressed in Xenopus laevis oocytes. 5-HT (1 μM)-evoked currents recorded by two-electrode voltage-clamp technique were reversibly inhibited by menthol in a concentration-dependent (IC50 = 163 μM) manner. The effects of menthol developed gradually, reaching a steady-state level within 10-15 minutes and did not involve G-proteins, since GTPγS activity remained unaltered and the effect of menthol was not sensitive to pertussis toxin pretreatment. The actions of menthol were not stereoselective as (-), (+), and racemic menthol inhibited 5-HT3 receptor-mediated currents to the same extent. Menthol inhibition was not altered by intracellular 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid injections and transmembrane potential changes. The maximum inhibition observed for menthol was not reversed by increasing concentrations of 5-HT. Furthermore, specific binding of the 5-HT3 antagonist [(3)H]GR65630 was not altered in the presence of menthol (up to 1 mM), indicating that menthol acts as a noncompetitive antagonist of the 5-HT3 receptor. Finally, 5-HT3 receptor-mediated currents in acutely dissociated nodose ganglion neurons were also inhibited by menthol (100 μM). These data demonstrate that menthol, at pharmacologically relevant concentrations, is an allosteric inhibitor of 5-HT3 receptors.

  9. Reduced Mechanical Stretch Induces Enhanced Endothelin B Receptor-mediated Contractility via Activation of Focal Adhesion Kinase and Extra Cellular-regulated Kinase 1/2 in Cerebral Arteries from Rat

    DEFF Research Database (Denmark)

    Spray, Stine; Rasmussen, Marianne N P; Skovsted, Gry F

    2016-01-01

    Cerebral ischaemia results in enhanced endothelin B (ETB ) receptor-mediated contraction and receptor protein expression in the affected cerebrovascular smooth muscle cells (SMC). Organ culture of cerebral arteries is a method to induce similar alterations in ETB receptor expression. We hypothesize...... expression to SMC expression and 2) an increased calcium sensitivity of the SMCs due to an increased expression of the calcium channel transient receptor potential canonical 1. Collectively, our results present a possible mechanism linking lack of vessel wall stretch/tension to changes in ETB receptor...

  10. Isolated NMDA receptor-mediated synaptic responses express both LTP and LTD.

    Science.gov (United States)

    Xie, X; Berger, T W; Barrionuevo, G

    1992-04-01

    1. The possibility of use-dependent, long-lasting modifications of pharmacologically isolated N-methyl-D-aspartate (NMDA) receptor-mediated synaptic transmission was examined by intracellular recordings from granule cells of the hippocampal dentate gyrus in vitro. In the presence of the non-NMDA receptor antagonist 6-cyano-7-nitroquinaxaline-2,3-dione (CNQX, 10 microM) robust, long-term potentiation (LTP) of NMDA receptor-mediated synaptic potentials was induced by brief, high (50 Hz) and lower (10 Hz) frequency tetanic stimuli of glutamatergic afferents (60 +/- 6%, n = 8, P less than 0.001 and 43 +/- 12%, n = 3, P less than 0.05, respectively). 2. Hyperpolarization of granule cell membrane potential to -100 mV during 50-Hz tetanic stimuli reversibly blocked the induction of LTP (-6 +/- 2%, n = 6, P greater than 0.05) indicating that simultaneous activation of pre- and postsynaptic elements is a prerequisite for potentiation of NMDA receptor-mediated synaptic transmission. In contrast, hyperpolarization of the granule cell membrane potential to -100 mV during 10-Hz tetanic stimuli resulted in long-term depression (LTD) of NMDA receptor-mediated synaptic potentials (-34 +/- 8%, n = 8, P less than 0.01). 3. We also studied the role of [Ca2+]i in the induction of LTP and LTD of NMDA receptor-mediated synaptic responses. Before tetanization, [Ca2+]i was buffered by iontophoretic injections of bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA). BAPTA completely blocked the induction of LTP (3 +/- 5%, n = 13) and partially blocked LTD (-14.8 +/- 6%, n = 10).(ABSTRACT TRUNCATED AT 250 WORDS)

  11. THIP, a hypnotic and antinociceptive drug, enhances a tonic GABAA receptor mediated conductance in mouse neocortex

    DEFF Research Database (Denmark)

    Drasbek, Kim Ryun; Jensen, Kimmo

    2006-01-01

    its cellular actions in the neocortex are uncertain, we studied the effects of THIP on neurons in slices of frontoparietal neocortex of 13- to 19-day-old (P13-19) mice. Using whole-cell patch-clamp recordings, we found that the clinically relevant THIP concentration of 1 μM induced a robust tonic GABA...... suggest that THIP activates an extrasynaptic GABA(A) receptor-mediated conductance in the neocortex, which may alter the cortical network activity....

  12. Glutamate receptor-mediated toxicity in optic nerve oligodendrocytes

    Science.gov (United States)

    Matute, Carlos; Sánchez-Gómez, M. Victoria; Martínez-Millán, Luis; Miledi, Ricardo

    1997-01-01

    In cultured oligodendrocytes isolated from perinatal rat optic nerves, we have analyzed the expression of ionotropic glutamate receptor subunits as well as the effect of the activation of these receptors on oligodendrocyte viability. Reverse transcription–PCR, in combination with immunocytochemistry, demonstrated that most oligodendrocytes differentiated in vitro express the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluR3 and GluR4 and the kainate receptor subunits GluR6, GluR7, KA1 and KA2. Acute and chronic exposure to kainate caused extensive oligodendrocyte death in culture. This effect was partially prevented by the AMPA receptor antagonist GYKI 52466 and was completely abolished by the non-N-methyl-d-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), suggesting that both AMPA and kainate receptors mediate the observed kainate toxicity. Furthermore, chronic application of kainate to optic nerves in vivo resulted in massive oligodendrocyte death which, as in vitro, could be prevented by coinfusion of the toxin with CNQX. These findings suggest that excessive activation of the ionotropic glutamate receptors expressed by oligodendrocytes may act as a negative regulator of the size of this cell population. PMID:9238063

  13. Fcγ receptor-mediated inflammation inhibits axon regeneration.

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    Gang Zhang

    Full Text Available Anti-glycan/ganglioside antibodies are the most common immune effectors found in patients with Guillain-Barré Syndrome, which is a peripheral autoimmune neuropathy. We previously reported that disease-relevant anti-glycan autoantibodies inhibited axon regeneration, which echo the clinical association of these antibodies and poor recovery in Guillain-Barré Syndrome. However, the specific molecular and cellular elements involved in this antibody-mediated inhibition of axon regeneration are not previously defined. This study examined the role of Fcγ receptors and macrophages in the antibody-mediated inhibition of axon regeneration. A well characterized antibody passive transfer sciatic nerve crush and transplant models were used to study the anti-ganglioside antibody-mediated inhibition of axon regeneration in wild type and various mutant and transgenic mice with altered expression of specific Fcγ receptors and macrophage/microglia populations. Outcome measures included behavior, electrophysiology, morphometry, immunocytochemistry, quantitative real-time PCR, and western blotting. We demonstrate that the presence of autoantibodies, directed against neuronal/axonal cell surface gangliosides, in the injured mammalian peripheral nerves switch the proregenerative inflammatory environment to growth inhibitory milieu by engaging specific activating Fcγ receptors on recruited monocyte-derived macrophages to cause severe inhibition of axon regeneration. Our data demonstrate that the antibody orchestrated Fcγ receptor-mediated switch in inflammation is one mechanism underlying inhibition of axon regeneration. These findings have clinical implications for nerve repair and recovery in antibody-mediated immune neuropathies. Our results add to the complexity of axon regeneration in injured peripheral and central nervous systems as adverse effects of B cells and autoantibodies on neural injury and repair are increasingly recognized.

  14. Thromboxane A2 receptor-mediated release of matrix metalloproteinase-1 (MMP-1) induces expression of monocyte chemoattractant protein-1 (MCP-1) by activation of protease-activated receptor 2 (PAR2) in A549 human lung adenocarcinoma cells.

    Science.gov (United States)

    Li, Xiuling; Tai, Hsin-Hsiung

    2014-08-01

    Matrix metalloproteinases (MMPs) and monocyte chemoattractant protein-1 (MCP-1, CCL2) are known to be upregulated in many tumors. Their roles in tumor invasion and metastasis are being uncovered. How they are related to each other and involved in tumor progression remains to be determined. Earlier it was reported that I-BOP-initiated activation of thromboxane A2 receptor (TP) induced the release of MMP-1, MMP-3, and MMP-9 from lung cancer A549 cells overexpressing TPα (A549-TPα). Herein it was found that MMP-1, but not MMP-3 or MMP-9, induced the expression of MCP-1 in A549 cells. Conditioned medium (CM) from I-BOP activated, MMP-1 siRNA pretreated A549-TPα cells induced greatly attenuated expression of MCP-1 in A549 cells indicating that MMP-1 in the CM contributed significantly to the expression of MCP-1. MMP-1 was shown to activate protease-activated receptor 2 (PAR2) instead of commonly assumed PAR1 to increase the expression of MCP-1 in A549 cells. This conclusion was reached from the following findings: (1) expression of MCP-1 induced by trypsin, a PAR2 agonist, and also PAR2 agonist peptide, was inhibited by a PAR2 antagonist; (2) expression of MCP-1 induced by MMP-1 and by CM from I-BOP activated A549-TPα cells was blocked by a PAR2 antagonist but not by other PAR antagonists; (3) expression of MCP-1 induced by MMP-1 and by CM from I-BOP activated A549-TPα cells was attenuated significantly by pretreatment of cells with PAR2-siRNA. These results suggest that PAR2 is a novel MMP-1 target mediating MMP-1-induced signals in A549 lung cancer cells.

  15. Bim regulates B-cell receptor-mediated apoptosis in the presence of CD40 signaling in CD40-pre-activated splenic B cells differentiating into plasma cells.

    Science.gov (United States)

    Gao, Yuanyuan; Kazama, Hirotaka; Yonehara, Shin

    2012-05-01

    B-cell receptor (BCR)-mediated apoptosis is critical for B-cell development and homeostasis. CD40 signaling has been shown to protect immature or mature B cells from BCR-mediated apoptosis. In this study, to understand the fate of CD40-pre-activated splenic B cells stimulated by BCR engagement in the presence of CD40 signaling, murine splenic B cells were cultured with anti-Igκ and anti-CD40 antibodies after pre-activation with anti-CD40 antibody. We found that apoptosis was induced in the cultured B cells even in the presence of CD40 signaling during the 3-4 days cultivation. We detected up-regulation of Bim expression followed by Bax activation in this apoptotic process and cessation of the apoptosis in Bim-deficient B cells, indicating that Bim is a key regulator of the BCR-mediated apoptosis in the presence of CD40 signaling in CD40-pre-activated B cells. Importantly, this BCR-mediated apoptosis in CD40-pre-activated B cells was shown to be induced at the initiation of plasma cell differentiation at around the preplasmablast stage, and Bim-deficient B cells cultured under these conditions differentiated into plasma cells. Additionally, transforming growth factor-β was found to protect CD40-pre-activated B cells from BCR-mediated apoptosis in the presence of CD40 signaling. Our identified BCR-mediated apoptosis, which is unpreventable by CD40 signaling, suggests a potential mechanism that regulates the elimination of peripheral B cells, which should be derived from nonspecific T-dependent activation of bystander B cells and continuous stimulation with antigens including self-antigens in the presence of T cell help through CD40.

  16. Melanocortin MC(4) receptor-mediated feeding and grooming in rodents.

    Science.gov (United States)

    Mul, Joram D; Spruijt, Berry M; Brakkee, Jan H; Adan, Roger A H

    2013-11-01

    Decades ago it was recognized that the pharmacological profile of melanocortin ligands that stimulated grooming behavior in rats was strikingly similar to that of Xenopus laevis melanophore pigment dispersion. After cloning of the melanocortin MC1 receptor, expressed in melanocytes, and the melanocortin MC4 receptor, expressed mainly in brain, the pharmacological profiles of these receptors appeared to be very similar and it was demonstrated that these receptors mediate melanocortin-induced pigmentation and grooming respectively. Grooming is a low priority behavior that is concerned with care of body surface. Activation of central melanocortin MC4 receptors is also associated with meal termination, and continued postprandial stimulation of melanocortin MC4 receptors may stimulate natural postprandial grooming behavior as part of the behavioral satiety sequence. Indeed, melanocortins fail to suppress food intake or induce grooming behavior in melanocortin MC4 receptor-deficient rats. This review will focus on how melanocortins affect grooming behavior through the melanocortin MC4 receptor, and how melanocortin MC4 receptors mediate feeding behavior. This review also illustrates how melanocortins were the most likely candidates to mediate grooming and feeding based on the natural behaviors they induced.

  17. Folate receptor mediated intracellular protein delivery using PLL-PEG-FOL conjugate.

    Science.gov (United States)

    Hwa Kim, Sun; Hoon Jeong, Ji; Joe, Cheol O; Gwan Park, Tae

    2005-04-18

    To develop a receptor-mediated intracellular delivery system that can transport therapeutic proteins or other bioactive macromolecules into a specific cell, a di-block copolymer conjugate, poly(L-lysine)-poly(ethylene glycol)-folate (PLL-PEG-FOL), was synthesized. The PLL-PEG-FOL conjugate was physically complexed with fluorescein isothiocyanate conjugated bovine serum albumin (FITC-BSA) in an aqueous phase by ionic interactions. Cellular uptake of PLL-PEG-FOL/FITC-BSA complexes was greatly enhanced against a folate receptor over-expressing cell line (KB cells) compared to a folate receptor deficient cell line (A549 cells). The presence of an excess amount of free folate (1 mM) in the medium inhibited the intracellular delivery of PLL-PEG-FOL/FITC-BSA complexes. This suggests that the enhanced cellular uptake of FITC-BSA by KB cells in a specific manner was attributed to folate receptor-mediated endocytosis of the complexes having folate moieties on the surface. The PLL-PEG-FOL di-block copolymer could be potentially applied for intracellular delivery of a wide range of other biological active agents that have negative charges on the surface.

  18. Store-Operated Ca2+ Entry (SOCE) and Purinergic Receptor-Mediated Ca2+ Homeostasis in Murine bv2 Microglia Cells: Early Cellular Responses to ATP-Mediated Microglia Activation

    Science.gov (United States)

    Gilbert, Daniel F.; Stebbing, Martin J.; Kuenzel, Katharina; Murphy, Robyn M.; Zacharewicz, Evelyn; Buttgereit, Andreas; Stokes, Leanne; Adams, David J.; Friedrich, Oliver

    2016-01-01

    Microglia activation is a neuroinflammatory response to parenchymal damage with release of intracellular metabolites, e.g., purines, and signaling molecules from damaged cells. Extracellular purines can elicit Ca2+-mediated microglia activation involving P2X/P2Y receptors with metabotropic (P2Y) and ionotropic (P2X) cell signaling in target cells. Such microglia activation results in increased phagocytic activity, activation of their inflammasome and release of cytokines to sustain neuroinflammatory (so-called M1/M2 polarization). ATP-induced activation of ionotropic P2X4 and P2X7 receptors differentially induces receptor-operated Ca2+ entry (ROCE). Although store-operated Ca2+ entry (SOCE) was identified to modulate ROCE in primary microglia, its existence and role in one of the most common murine microglia cell line, BV2, is unknown. To dissect SOCE from ROCE in BV2 cells, we applied high-resolution multiphoton Ca2+ imaging. After depleting internal Ca2+ stores, SOCE was clearly detectable. High ATP concentrations (1 mM) elicited sustained increases in intracellular [Ca2+]i whereas lower concentrations (≤100 μM) also induced Ca2+ oscillations. These differential responses were assigned to P2X7 and P2X4 activation, respectively. Pharmacologically inhibiting P2Y and P2X responses did not affect SOCE, and in fact, P2Y-responses were barely detectable in BV2 cells. STIM1S content was significantly upregulated by 1 mM ATP. As P2X-mediated Ca2+ oscillations were rare events in single cells, we implemented a high-content screening approach that allows to record Ca2+ signal patterns from a large number of individual cells at lower optical resolution. Using automated classifier analysis, several drugs (minocycline, U73122, U73343, wortmannin, LY294002, AZ10606120) were tested on their profile to act on Ca2+ oscillations (P2X4) and sustained [Ca2+]i increases. We demonstrate specific drug effects on purinergic Ca2+ pathways and provide new pharmacological insights into

  19. STORE-OPERATED CA2+ ENTRY (SOCE AND PURINERGIC RECEPTOR-MEDIATED CA2+ HOMEOSTASIS IN MURINE BV2 MICROGLIA CELLS: EARLY CELLULAR RESPONSES TO ATP-MEDIATED MICROGLIA ACTIVATION

    Directory of Open Access Journals (Sweden)

    Daniel F. Gilbert

    2016-10-01

    Full Text Available Microglia activation is a neuro-inflammatory response to parenchymal damage with release of intracellular metabolites, e.g. purines, and signaling molecules from damaged cells. Extracellular purines can elicit Ca2+-mediated microglia activation involving P2X/P2Y receptors with metabotropic (P2Y and ionotropic (P2X cell signaling in target cells. Such microglia activation results in increased phagocytic activity, activation of their inflammasome and release of cytokines to sustain neuro-inflammation (so-called M1/M2 polarization. ATP-induced activation of ionotropic P2X4 and P2X7 receptors differentially induce receptor-operated Ca2+ entry (ROCE. Although store-operated Ca2+ entry (SOCE was identified to modulate ROCE in primary microglia, its existence and role in one of the most common murine microglia cell line, BV2, is unknown. To dissect SOCE from ROCE in BV2 cells, we applied high-resolution multiphoton Ca2+ imaging. After depleting internal Ca2+ stores, SOCE was clearly detectable. High ATP concentrations (1 mM elicited sustained increases in intracellular [Ca2+]i whereas lower concentrations (≤100 µM also induced Ca2+ oscillations. These differential responses were assigned to P2X7 and P2X4 activation, respectively. Pharmacologically inhibiting P2Y and P2X responses did not affect SOCE, and in fact, P2Y-responses were barely detectable in BV2 cells. STIM1S content was significantly upregulated by 1 mM ATP. As P2X-mediated Ca2+ oscillations were rare events in single cells, we implemented a high-content screening approach that allows to record Ca2+ signal patterns from a large number of individual cells at lower optical resolution. Using automated classifier analysis, several drugs (minocycline, U73122, U73343, wortmannin, LY294002, AZ10606120 were tested on their profile to act on Ca2+ oscillations (P2X4 and sustained [Ca2+]i increases. We demonstrate specific drug effects on purinergic Ca2+ pathways and provide new pharmacological

  20. Active Calcium/Calmodulin-Dependent Protein Kinase II (CaMKII Regulates NMDA Receptor Mediated Postischemic Long-Term Potentiation (i-LTP by Promoting the Interaction between CaMKII and NMDA Receptors in Ischemia

    Directory of Open Access Journals (Sweden)

    Ning Wang

    2014-01-01

    Full Text Available Active calcium/calmodulin-dependent protein kinase II (CaMKII has been reported to take a critical role in the induction of long-term potentiation (LTP. Changes in CaMKII activity were detected in various ischemia models. It is tempting to know whether and how CaMKII takes a role in NMDA receptor (NMDAR-mediated postischemic long-term potentiation (NMDA i-LTP. Here, we monitored changes in NMDAR-mediated field excitatory postsynaptic potentials (NMDA fEPSPs at different time points following ischemia onset in vitro oxygen and glucose deprivation (OGD ischemia model. We found that 10 min OGD treatment induced significant i-LTP in NMDA fEPSPs, whereas shorter (3 min or longer (25 min OGD treatment failed to induce prominent NMDA i-LTP. CaMKII activity or CaMKII autophosphorylation displays a similar bifurcated trend at different time points following onset of ischemia both in vitro OGD or in vivo photothrombotic lesion (PT models, suggesting a correlation of increased CaMKII activity or CaMKII autophosphorylation with NMDA i-LTP. Disturbing the association between CaMKII and GluN2B subunit of NMDARs with short cell-permeable peptides Tat-GluN2B reversed NMDA i-LTP induced by OGD treatment. The results provide support to a notion that increased interaction between NMDAR and CaMKII following ischemia-induced increased CaMKII activity and autophosphorylation is essential for induction of NMDA i-LTP.

  1. microRNA-146a inhibits G protein-coupled receptor-mediated activation of NF-κB by targeting CARD10 and COPS8 in gastric cancer

    Directory of Open Access Journals (Sweden)

    Crone Stephanie

    2012-09-01

    Full Text Available Abstract Background Gastric cancer is the second most common cause of cancer-related death in the world. Inflammatory signals originating from gastric cancer cells are important for recruiting inflammatory cells and regulation of metastasis of gastric cancer. Several microRNAs (miRNA have been shown to be involved in development and progression of gastric cancer. miRNA-146a (miR-146a is a modulator of inflammatory signals, but little is known about its importance in gastric cancer. We therefore wanted to identify targets of miR-146a in gastric cancer and examine its biological roles. Results The expression of miR-146a was evaluated by quantitative PCR (qPCR and found up-regulated in the gastrin knockout mice, a mouse model of gastric cancer, and in 73% of investigated human gastric adenocarcinomas. Expression of miR-146a by gastric cancer cells was confirmed by in situ hybridization. Global analysis of changes in mRNA levels after miR-146a transfection identified two transcripts, caspase recruitment domain-containing protein 10 (CARD10 and COP9 signalosome complex subunit 8 (COPS8, as new miR-146a targets. qPCR, Western blotting and luciferase assays confirmed these transcripts as direct miR-146a targets. CARD10 and COPS8 were shown to be part of the G protein-coupled receptor (GPCR pathway of nuclear factor-kappaB (NF-kappaB activation. Lysophosphatidic acid (LPA induces NF-kappaB activation via this pathway and over-expression of miR-146a inhibited LPA-induced NF-kappaB activation, reduced LPA-induced expression of tumor-promoting cytokines and growth factors and inhibited monocyte attraction. Conclusions miR-146a expression is up-regulated in a majority of gastric cancers where it targets CARD10 and COPS8, inhibiting GPCR-mediated activation of NF-kappaB, thus reducing expression of NF-kappaB-regulated tumor-promoting cytokines and growth factors. By targeting components of several NF-kappaB-activating pathways, miR-146a is a key component in

  2. GABAA Receptor-Mediated Bidirectional Control of Synaptic Activity, Intracellular Ca2+, Cerebral Blood Flow, and Oxygen Consumption in Mouse Somatosensory Cortex In Vivo

    DEFF Research Database (Denmark)

    Jessen, Sanne Barsballe; Brazhe, Alexey; Lind, Barbara Lykke

    2015-01-01

    Neural activity regulates local increases in cerebral blood flow (ΔCBF) and the cortical metabolic rate of oxygen (ΔCMRO2) that constitutes the basis of BOLD functional neuroimaging signals. Glutamate signaling plays a key role in brain vascular and metabolic control; however, the modulatory effe...... selectively gate and amplify transient low-frequency somatosensory inputs, filter out high-frequency inputs, and enhance vascular and metabolic responses that are likely to be reflected in BOLD functional neuroimaging signals....

  3. Caffeine inhibits the activation of hepatic stellate cells induced by acetaldehyde via adenosine A2A receptor mediated by the cAMP/PKA/SRC/ERK1/2/P38 MAPK signal pathway.

    Directory of Open Access Journals (Sweden)

    He Wang

    Full Text Available Hepatic stellate cell (HSC activation is an essential event during alcoholic liver fibrosis. Evidence suggests that adenosine aggravates liver fibrosis via the adenosine A2A receptor (A2AR. Caffeine, which is being widely consumed during daily life, inhibits the action of adenosine. In this study, we attempted to validate the hypothesis that caffeine influences acetaldehyde-induced HSC activation by acting on A2AR. Acetaldehyde at 50, 100, 200, and 400 μM significantly increased HSC-T6 cells proliferation, and cell proliferation reached a maximum at 48 h after exposure to 200 μM acetaldehyde. Caffeine and the A2AR antagonist ZM241385 decreased the cell viability and inhibited the expression of procollagen type I and type III in acetaldehyde-induced HSC-T6 cells. In addition, the inhibitory effect of caffeine on the expression of procollagen type I was regulated by A2AR-mediated signal pathway involving cAMP, PKA, SRC, and ERK1/2. Interestingly, caffeine's inhibitory effect on the expression of procollagen type III may depend upon the A2AR-mediated P38 MAPK-dependent pathway.Caffeine significantly inhibited acetaldehyde-induced HSC-T6 cells activation by distinct A2AR mediated signal pathway via inhibition of cAMP-PKA-SRC-ERK1/2 for procollagen type I and via P38 MAPK for procollagen type III.

  4. Myelin-associated glycoprotein modulates apoptosis of motoneurons during early postnatal development via NgR/p75(NTR) receptor-mediated activation of RhoA signaling pathways.

    Science.gov (United States)

    Palandri, A; Salvador, V R; Wojnacki, J; Vivinetto, A L; Schnaar, R L; Lopez, P H H

    2015-09-03

    Myelin-associated glycoprotein (MAG) is a minor constituent of nervous system myelin, selectively expressed on the periaxonal myelin wrap. By engaging multiple axonal receptors, including Nogo-receptors (NgRs), MAG exerts a nurturing and protective effect the axons it ensheaths. Pharmacological activation of NgRs has a modulatory role on p75(NTR)-dependent postnatal apoptosis of motoneurons (MNs). However, it is not clear whether this reflects a physiological role of NgRs in MN development. NgRs are part of a multimeric receptor complex, which includes p75(NTR), Lingo-1 and gangliosides. Upon ligand binding, this multimeric complex activates RhoA/ROCK signaling in a p75(NTR)-dependent manner. The aim of this study was to analyze a possible modulatory role of MAG on MN apoptosis during postnatal development. A time course study showed that Mag-null mice suffer a loss of MNs during the first postnatal week. Also, these mice exhibited increased susceptibility in an animal model of p75(NTR)-dependent MN apoptosis induced by nerve-crush injury, which was prevented by treatment with a soluble form of MAG (MAG-Fc). The protective role of MAG was confirmed in in vitro models of p75(NTR)-dependent MN apoptosis using the MN1 cell line and primary cultures. Lentiviral expression of shRNA sequences targeting NgRs on these cells abolished protection by MAG-Fc. Analysis of RhoA activity using a FRET-based RhoA biosensor showed that MAG-Fc activates RhoA. Pharmacological inhibition of p75(NTR)/RhoA/ROCK pathway, or overexpression of a p75(NTR) mutant unable to activate RhoA, completely blocked MAG-Fc protection against apoptosis. The role of RhoA/ROCK signaling was further confirmed in the nerve-crush model, where pretreatment with ROCK inhibitor Y-27632 blocked the pro-survival effect of MAG-Fc. These findings identify a new protective role of MAG as a modulator of apoptosis of MNs during postnatal development by a mechanism involving the p75(NTR)/RhoA/ROCK signaling pathway

  5. Quercetin modulates toll-like receptor-mediated protein kinase signaling pathways in oxLDL-challenged human PBMCs and regulates TLR-activated atherosclerotic inflammation in hypercholesterolemic rats.

    Science.gov (United States)

    Bhaskar, Shobha; Helen, A

    2016-12-01

    Toll-like receptors (TLRs) are pattern recognition receptors that have a unique and essential function in innate immunity. The effect of quercetin on TLR-mediated downstream signaling mechanism and its effect on TLR-mediated MAP kinase and Akt pathways were studied in oxLDL-stimulated hPBMCs using specific inhibitors. The pretreatment of hPBMCs with specific TLR inhibitor, CLI-095, decreased the NF-κB nuclear translocation and TNF-α release by oxLDL. When the cells treated with inhibitor and quercetin together, the inhibition was more effective. The specific inhibitor for p38 MAPK, SB203580, reduced the phosphorylated p38 level and decreased the NF-κB activation and TNF-α release by oxLDL-challenged hPBMCs. This inhibitor showed enhanced inhibition when treated with quercetin together. The inhibitors for ERK1/2, PD98059, and for JNK, SP606125, also showed inhibitory effect on NF-κB activation and TNF-α release by oxLDL-simulated hPBMCs. Quercetin supplementation enhanced the inhibition of nuclear translocation of NF-κB and the release of cytokines. TLR4 inhibition study confirmed the downstream signaling mechanism mediated by NF-κB which is involved in the oxLDL-induced inflammatory response, and quercetin suppresses the cytokine, TNF-α release by modulating TLR-NF-κB signaling pathway. In addition to NF-κB signaling pathway, inflammation induced by oxLDL was also related to the activation of p38MAPK, ERK1/2 and JNK, and Akt pathways, and the protective effect of quercetin may be also related to the inhibition of activation of these pathways. Quercetin significantly downregulated the elevated mRNA expression of TLRs and cytokine TNF-α in HCD-fed atherosclerotic rats in vivo. As quercetin possesses inhibition on both TLR-NF-κB signaling pathway and TLR-mediated MAPK pathway, it is evident that it can be used as a therapeutic agent to ameliorate atherosclerotic inflammation. Since quercetin is the major flavonoid and forms the backbone of many other

  6. Nfkb1 activation by the E26 transformation-specific transcription factors PU.1 and Spi-B promotes Toll-like receptor-mediated splenic B cell proliferation.

    Science.gov (United States)

    Li, Stephen K H; Abbas, Ali K; Solomon, Lauren A; Groux, Gaëlle M N; DeKoter, Rodney P

    2015-05-01

    Generation of antibodies against T-independent and T-dependent antigens requires Toll-like receptor (TLR) engagement on B cells for efficient responses. However, the regulation of TLR expression and responses in B cells is not well understood. PU.1 and Spi-B (encoded by Sfpi1 and Spib, respectively) are transcription factors of the E26 transformation-specific (ETS) family and are important for B cell development and function. It was found that B cells from mice knocked out for Spi-B and heterozygous for PU.1 (Sfpi1(+/-) Spib(-/-) [PUB] mice) proliferated poorly in response to TLR ligands compared to wild-type (WT) B cells. The NF-κB family member p50 (encoded by Nfkb1) is required for lipopolysaccharide (LPS) responsiveness in mice. PUB B cells expressed reduced Nfkb1 mRNA transcripts and p50 protein. The Nfkb1 promoter was regulated directly by PU.1 and Spi-B, as shown by reporter assays and chromatin immunoprecipitation analysis. Occupancy of the Nfkb1 promoter by PU.1 was reduced in PUB B cells compared to that in WT B cells. Finally, infection of PUB B cells with a retroviral vector encoding p50 substantially restored proliferation in response to LPS. We conclude that Nfkb1 transcriptional activation by PU.1 and Spi-B promotes TLR-mediated B cell proliferation.

  7. Interleukin 1 (IL-1) type I receptors mediate activation of rat hypothalamus-pituitary-adrenal axis and interleukin 6 production as shown by receptor type selective deletion mutants of IL-1beta.

    Science.gov (United States)

    Van Dam, A M; Malinowsky, D; Lenczowski, M J; Bartfai, T; Tilders, F J

    1998-06-01

    The cytokine interleukin 1 (IL-1) plays an important role in the activation of the hypothalamus-pituary-adrenal (HPA)-axis and interleukin 6 (IL-6) production during infection or inflammation. Which of the interleukin-1 receptor types mediates these effects is not known. To investigate this issue a pharmacological approach was chosen by using recently developed IL-1 receptor type selective ligands. Rats were given one of various doses of recombinant human IL-1beta (rhIL-1beta; 1 and 10 microg/kg) and of several IL-1beta mutants (DeltaSND, DeltaQGE and DeltaI; 1, 10 and 100 microg/kg), that differ in their affinities for the IL-1 type I receptor but have similar affinities for the IL-1 type II receptor. One hour after intravenous administration of rhIL-1beta or IL-1beta mutants, plasma levels of ACTH, corticosterone (cort) and IL-6 were measured. Doses of 1 and 10 microg/kg rhIL-1beta markedly elevated plasma levels of ACTH, cort and IL-6. However, 10-100-fold higher doses of IL-1beta mutants DeltaSND and DeltaQGE and at least 100-fold higher doses of DeltaI have to be administered to increase plasma levels of ACTH, cort and IL-6. The potency differences correlate with their respective affinity for the type I receptor but not with that of the IL-1 type II receptor. It is concluded that IL-1beta induced ACTH, cort and IL-6 production is mediated by interleukin 1 type I receptors.

  8. The N-terminal extracellular domain 23-60 of the calcitonin receptor-like receptor in chimeras with the parathyroid hormone receptor mediates association with receptor activity-modifying protein 1.

    Science.gov (United States)

    Ittner, Lars M; Koller, Daniela; Muff, Roman; Fischer, Jan A; Born, Walter

    2005-04-19

    The calcitonin receptor-like receptor (CLR) requires the associated receptor activity-modifying protein (RAMP)1 to reveal a calcitonin gene-related peptide (CGRP) receptor. Here, the subdomain of the CLR that associates with RAMP1 has been identified in chimeras between the CLR and the parathyroid hormone (PTH) receptor 1 (PTHR). The PTHR alone does not interact with RAMP1. RAMP1 requires the CLR for its transport to the cell surface. Thus, receptor-dependent RAMP1 delivery to the plasma membrane and coimmunoprecipitation from the cell surface were used as measures for receptor/RAMP1 interaction. Several chimeric CLR-PTHR included the N-terminal amino acids 23-60 of the CLR transported RAMP1 to the surface of COS-7 cells much like the intact CLR. Moreover, RAMP1 coimmunoprecipitated with these receptors from the cell surface. A CLR deletion mutant, consisting of the N-terminal extracellular domain, the first transmembrane domain, and the C-terminal intracellular region, revealed the same results. Cyclic AMP was stimulated by CGRP in CLR/RAMP1 expressing cells (58 +/- 19-fold, EC(50) = 0.12 +/- 0.03 nM) and by PTH-related protein in cells expressing the PTHR (50 +/- 10-fold, EC(50) = 0.25 +/- 0.03 nM) or a PTHR with the N-terminal amino acids 23-60 of the CLR (23 +/- 5-fold, EC(50) > 1000 nM). Other chimeric CLR-PTHR were inactive. In conclusion, structural elements in the extreme N-terminus of the CLR between amino acids 23-60 are required and sufficient for CLR/RAMP1 cotransport to the plasma membrane and heterodimerization.

  9. Follicle-stimulating hormone receptor-mediated uptake of sup 45 Ca sup 2+ by proteoliposomes and cultured rat sertoli cells: Evidence for involvement of voltage-activated and voltage-independent calcium channels

    Energy Technology Data Exchange (ETDEWEB)

    Grasso, P.; Reichert, L.E. Jr. (Albany Medical College, NY (USA))

    1989-12-01

    We have previously reported incorporation into liposomes of Triton X-100-solubilized FSH receptor-G-protein complexes derived from purified bovine calf testis membranes. In the present study we have used this model system to show that FSH induces flux of 45Ca2+ into such proteoliposomes in a hormone-specific concentration-dependent manner. FSH, inactivated by boiling, had no stimulatory effect on 45Ca2+ flux, nor did isolated alpha- or beta-subunits of FSH. Addition of GTP (or its analogs 5'-guanylylimidodiphosphate and guanosine-5'-O-(3-thiotriphosphate)) or sodium fluoride (in the presence or absence of GTP or its analogs) failed to induce 45Ca2+ flux into proteoliposomes, suggesting that the uptake of 45Ca2+ was receptor, and not G-protein, related. Voltage-independent (ruthenium red and gadolinium chloride) and voltage-activated (methyoxyverapamil and nifedipine) calcium channel-blocking agents reduced FSH-stimulated 45Ca2+ flux into proteoliposomes to control levels. FSH also induced uptake of 45Ca2+ by cultured rat Sertoli cells. Ruthenium red and gadolinium chloride had no effect on basal levels of 45Ca2+ uptake or estradiol secretion by cultured rat Sertoli cells, nor did methoxyverapamil or nifedipine. All four calcium channel blockers, however, were able to reduce FSH-induced 45Ca2+ uptake to basal levels and FSH-stimulated conversion of androstenedione to estradiol by up to 50%, indicating an involvement of Ca2+ in FSH-stimulated steroidogenesis. Our results suggest that the well documented changes in intracellular calcium levels consequent to FSH binding may be due, at least in part, to an influx of calcium through FSH receptor-regulated calcium channels.

  10. Extracellular acidosis impairs P2Y receptor-mediated Ca(2+) signalling and migration of microglia.

    Science.gov (United States)

    Langfelder, Antonia; Okonji, Emeka; Deca, Diana; Wei, Wei-Chun; Glitsch, Maike D

    2015-04-01

    Microglia are the resident macrophage and immune cell of the brain and are critically involved in combating disease and assaults on the brain. Virtually all brain pathologies are accompanied by acidosis of the interstitial fluid, meaning that microglia are exposed to an acidic environment. However, little is known about how extracellular acidosis impacts on microglial function. The activity of microglia is tightly controlled by 'on' and 'off' signals, the presence or absence of which results in generation of distinct phenotypes in microglia. Activation of G protein coupled purinergic (P2Y) receptors triggers a number of distinct behaviours in microglia, including activation, migration, and phagocytosis. Using pharmacological tools and fluorescence imaging of the murine cerebellar microglia cell line C8B4, we show that extracellular acidosis interferes with P2Y receptor-mediated Ca(2+) signalling in these cells. Distinct P2Y receptors give rise to signature intracellular Ca(2+) signals, and Ca(2+) release from stores and Ca(2+) influx are differentially affected by acidotic conditions: Ca(2+) release is virtually unaffected, whereas Ca(2+) influx, mediated at least in part by store-operated Ca(2+) channels, is profoundly inhibited. Furthermore, P2Y1 and P2Y6-mediated stimulation of migration is inhibited under conditions of extracellular acidosis, whereas basal migration independent of P2Y receptor activation is not. Taken together, our results demonstrate that an acidic microenvironment impacts on P2Y receptor-mediated Ca(2+) signalling, thereby influencing microglial responses and responsiveness to extracellular signals. This may result in altered behaviour of microglia under pathological conditions compared with microglial responses in healthy tissue. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Current injection and receptor-mediated excitation produce similar maximal firing rates in hypoglossal motoneurons.

    Science.gov (United States)

    Wakefield, Hilary E; Fregosi, Ralph F; Fuglevand, Andrew J

    2016-03-01

    The maximum firing rates of motoneurons (MNs), activated in response to synaptic drive, appear to be much lower than that elicited by current injection. It could be that the decrease in input resistance associated with increased synaptic activity (but not current injection) might blunt overall changes in membrane depolarization and thereby limit spike-frequency output. To test this idea, we recorded, in the same cells, maximal firing responses to current injection and to synaptic activation. We prepared 300 μm medullary slices in neonatal rats that contained hypoglossal MNs and used whole-cell patch-clamp electrophysiology to record their maximum firing rates in response to triangular-ramp current injections and to glutamate receptor-mediated excitation. Brief pressure pulses of high-concentration glutamate led to significant depolarization, high firing rates, and temporary cessation of spiking due to spike inactivation. In the same cells, we applied current clamp protocols that approximated the time course of membrane potential change associated with glutamate application and with peak current levels large enough to cause spike inactivation. Means (SD) of maximum firing rates obtained in response to glutamate application were nearly identical to those obtained in response to ramp current injection [glutamate 47.1 ± 12.0 impulses (imp)/s, current injection 47.5 ± 11.2 imp/s], even though input resistance was 40% less during glutamate application compared with current injection. Therefore, these data suggest that the reduction in input resistance associated with receptor-mediated excitation does not, by itself, limit the maximal firing rate responses in MNs.

  12. M2-like macrophages are responsible for collagen degradation through a mannose receptor-mediated pathway

    DEFF Research Database (Denmark)

    Madsen, Daniel H; Leonard, Daniel; Masedunskas, Andrius

    2013-01-01

    of the collagen receptors mannose receptor (Mrc1) and urokinase plasminogen activator receptor-associated protein (Endo180 and Mrc2) impaired this intracellular collagen degradation pathway. This study demonstrates the importance of receptor-mediated cellular uptake to collagen turnover in vivo and identifies......Tissue remodeling processes critically depend on the timely removal and remodeling of preexisting collagen scaffolds. Nevertheless, many aspects related to the turnover of this abundant extracellular matrix component in vivo are still incompletely understood. We therefore took advantage of recent...... advances in optical imaging to develop an assay to visualize collagen turnover in situ and identify cell types and molecules involved in this process. Collagen introduced into the dermis of mice underwent cellular endocytosis in a partially matrix metalloproteinase-dependent manner and was subsequently...

  13. AB318. SPR-45 Decentralization reduces nicotinic receptor-mediated canine bladder contractions in vitro

    Science.gov (United States)

    Salvadeo, Danielle M.; Frara, Nagat; Braverman, Alan S.; Barbe, Mary F.; Ruggieri, Michael R.

    2016-01-01

    had no significant inhibitory effect on DMPP, epibatidine or nicotine-induced contraction in any group. Conclusions Nicotinic receptors mediate contraction in sham, reinnervated and decentralized bladders. This nicotinic receptor-mediated contraction is decreased after decentralization. TTX does not block nicotinic receptor-mediated contractions, indicating that action potentials are not required to induce contraction. In sham-operated dog bladders, the nicotine-induced contraction is blocked by ATR, suggesting that these nicotinic receptors are located on cholinergic nerve terminals and induce the release of acetylcholine, which activates muscarinic receptors on the smooth muscle. Funding Source(s) NIH-NINDS NS070267

  14. Potentiation of NMDA receptor-mediated transmission in striatal cholinergic interneurons

    Directory of Open Access Journals (Sweden)

    Manfred eOswald

    2015-04-01

    Full Text Available Pauses in the tonic firing of striatal cholinergic interneurons (CINs emerge during reward-related learning in response to conditioning of a neutral cue. We have previously reported that augmenting the postsynaptic response to cortical afferents in CINs is coupled to the emergence of a cell-intrinsic afterhyperpolarisation (AHP underlying pauses in tonic activity. Here we investigated in a bihemispheric rat-brain slice preparation the mechanisms of synaptic plasticity of excitatory afferents to CINs and the association with changes in the AHP. We found that high frequency stimulation (HFS of commissural corticostriatal afferents from the contralateral hemisphere induced a robust long-term depression (LTD of postsynaptic potentials (PSP in CINs. Depression of the PSP of smaller magnitude and duration was observed in response to HFS of the ipsilateral white matter or cerebral cortex. In Mg2+-free solution HFS induced NMDA receptor-dependent potentiation of the PSP, evident in both the maximal slope and amplitude of the PSP. The increase in maximal slope corroborates previous findings, and was blocked by antagonism of either D1-like dopamine receptors with SCH23390 or D2-like dopamine receptors with sulpiride during HFS in Mg2+-free solution. Potentiation of the slower PSP amplitude component was due to augmentation of the NMDA receptor-mediated potential as this was completely reversed on subsequent application of the NMDA receptor antagonist AP5. HFS similarly potentiated NMDA receptor currents isolated by blockade of AMPA/kainate receptors with CNQX. The plasticity-induced increase in the slow PSP component was directly associated with an increase in the subsequent AHP. Thus plasticity of cortical afferent synapses is ideally suited to influence the cue-induced firing dynamics of CINs, particularly through potentiation of NMDA receptor-mediated synaptic transmission.

  15. Asialoglycoprotein receptor mediated hepatocyte targeting - strategies and applications.

    Science.gov (United States)

    D'Souza, Anisha A; Devarajan, Padma V

    2015-04-10

    Hepatocyte resident afflictions continue to affect the human population unabated. The asialoglycoprotein receptor (ASGPR) is primarily expressed on hepatocytes and minimally on extra-hepatic cells. This makes it specifically attractive for receptor-mediated drug delivery with minimum concerns of toxicity. ASGPR facilitates internalization by clathrin-mediated endocytosis and exhibits high affinity for carbohydrates specifically galactose, N-acetylgalactosamine and glucose. Isomeric forms of sugar, galactose density and branching, spatial geometry and galactose linkages are key factors influencing ligand-receptor binding. Popular ligands for ASGPR mediated targeting are carbohydrate polymers, arabinogalactan and pullulan. Other ligands include galactose-bearing glycoproteins, glycopeptides and galactose modified polymers and lipids. Drug-ligand conjugates provide a viable strategy; nevertheless ligand-anchored nanocarriers provide an attractive option for ASGPR targeted delivery and are widely explored. The present review details various ligands and nanocarriers exploited for ASGPR mediated delivery of drugs to hepatocytes. Nanocarrier properties affecting ASGPR mediated uptake are discussed at length. The review also highlights the clinical relevance of ASGPR mediated targeting and applications in diagnostics. ASGPR mediated hepatocyte targeting provides great promise for improved therapy of hepatic afflictions.

  16. Cerebellar vermis H₂ receptors mediate fear memory consolidation in mice.

    Science.gov (United States)

    Gianlorenço, A C L; Riboldi, A M; Silva-Marques, B; Mattioli, R

    2015-02-01

    Histaminergic fibers are present in the molecular and granular layers of the cerebellum and have a high density in the vermis and flocullus. Evidence supports that the cerebellar histaminergic system is involved in memory consolidation. Our recent study showed that histamine injections facilitate the retention of an inhibitory avoidance task, which was abolished by pretreatment with an H2 receptor antagonist. In the present study, we investigated the effects of intracerebellar post training injections of H1 and H2 receptor antagonists as well as the selective H2 receptor agonist on fear memory consolidation. The cerebellar vermi of male mice were implanted with guide cannulae, and after three days of recovery, the inhibitory avoidance test was performed. Immediately after a training session, animals received a microinjection of the following histaminergic drugs: experiment 1, saline or chlorpheniramine (0.016, 0.052 or 0.16 nmol); experiment 2, saline or ranitidine (0.57, 2.85 or 5.07 nmol); and experiment 3, saline or dimaprit (1, 2 or 4 nmol). Twenty-four hours later, a retention test was performed. The data were analyzed using one-way analysis of variance (ANOVA) and Duncan's tests. Animals microinjected with chlorpheniramine did not show any behavioral effects at the doses that we used. Intra-cerebellar injection of the H2 receptor antagonist ranitidine inhibited, while the selective H2 receptor agonist dimaprit facilitated, memory consolidation, suggesting that H2 receptors mediate memory consolidation in the inhibitory avoidance task in mice.

  17. Nonlinear pharmacokinetics of therapeutic proteins resulting from receptor mediated endocytosis.

    Science.gov (United States)

    Krippendorff, Ben-Fillippo; Kuester, Katharina; Kloft, Charlotte; Huisinga, Wilhelm

    2009-06-01

    Receptor mediated endocytosis (RME) plays a major role in the disposition of therapeutic protein drugs in the body. It is suspected to be a major source of nonlinear pharmacokinetic behavior observed in clinical pharmacokinetic data. So far, mostly empirical or semi-mechanistic approaches have been used to represent RME. A thorough understanding of the impact of the properties of the drug and of the receptor system on the resulting nonlinear disposition is still missing, as is how to best represent RME in pharmacokinetic models. In this article, we present a detailed mechanistic model of RME that explicitly takes into account receptor binding and trafficking inside the cell and that is used to derive reduced models of RME which retain a mechanistic interpretation. We find that RME can be described by an extended Michaelis-Menten model that accounts for both the distribution and the elimination aspect of RME. If the amount of drug in the receptor system is negligible a standard Michaelis-Menten model is capable of describing the elimination by RME. Notably, a receptor system can efficiently eliminate drug from the extracellular space even if the total number of receptors is small. We find that drug elimination by RME can result in substantial nonlinear pharmacokinetics. The extent of nonlinearity is higher for drug/receptor systems with higher receptor availability at the membrane, or faster internalization and degradation of extracellular drug. Our approach is exemplified for the epidermal growth factor receptor system.

  18. Histamine H3 receptor-mediated inhibition of noradrenaline release in the human brain.

    Science.gov (United States)

    Schlicker, E; Werthwein, S; Zentner, J

    1999-01-01

    Stimulation-evoked 3H-noradrenaline release in human cerebrocortical slices was inhibited by histamine (in a manner sensitive to clobenpropit) and by imetit, suggesting H3 receptor-mediated inhibition of noradrenaline release in human brain.

  19. Glucocorticoid Receptor-Mediated Repression of Pro-Inflammatory Genes in Rheumatoid Arthritis

    Science.gov (United States)

    2015-10-01

    1 AWARD NUMBER: W81XWH-14-1-0314 TITLE: Glucocorticoid Receptor-Mediated Repression of Pro-Inflammatory Genes in Rheumatoid Arthritis ...19 Sep 2015 4. TITLE AND SUBTITLE Glucocorticoid Receptor-Mediated Repression of Pro- Inflammatory Genes in Rheumatoid Arthritis 5a. CONTRACT NUMBER... arthritis (RA) patients rely on glucocorticoids (GCs) at some point during the disease. GCs signal through the GC receptor (GR), a transcription factor that

  20. Cellular mechanisms of the 5-HT7 receptor-mediated signaling.

    Science.gov (United States)

    Guseva, Daria; Wirth, Alexander; Ponimaskin, Evgeni

    2014-01-01

    Serotonin (5-hydroxytryptamine or 5-HT) is an important neurotransmitter regulating a wide range of physiological and pathological functions via activation of heterogeneously expressed 5-HT receptors. The 5-HT7 receptor is one of the most recently described members of the 5-HT receptor family. Functionally, 5-HT7 receptor is associated with a number of physiological and pathological responses, including serotonin-induced phase shifting of the circadian rhythm, control of memory as well as locomotor and exploratory activity. A large body of evidence indicates involvement of the 5-HT7 receptor in anxiety and depression, and recent studies suggest that 5-HT7 receptor can be highly relevant for the treatment of major depressive disorders. The 5-HT7 receptor is coupled to the stimulatory Gs-protein, and receptor stimulation results in activation of adenylyl cyclase (AC) leading to a rise of cAMP concentration. In addition, this receptor is coupled to the G12-protein to activate small GTPases of the Rho family. This review focuses on molecular mechanisms responsible for the 5-HT7 receptor-mediated signaling. We provide detailed overview of signaling cascades controlled and regulated by the 5-HT7 receptor and discuss the functional impact of 5-HT7 receptor for the regulation of different cellular and subcellular processes.

  1. Cellular mechanisms of the 5-HT7 receptor-mediated signaling

    Directory of Open Access Journals (Sweden)

    Daria eGuseva

    2014-10-01

    Full Text Available Serotonin (5-hydroxytryptamine or 5-HT is an important neurotransmitter regulating a wide range of physiological and pathological functions via activation of heterogeneously expressed 5-HT receptors. The 5-HT7 receptor is one of the most recently described members of the 5-HT receptor family. Functionally, 5-HT7 receptor is associated with a number of physiological and pathological responses, including serotonin-induced phase shifting of the circadian rhythm, control of memory as well as locomotor and exploratory activity. A large body of evidence indicates involvement of the 5-HT7 receptor in anxiety and depression, and recent studies suggest that 5-HT7 receptor can be highly relevant for the treatment of major depressive disorders. The 5-HT7 receptor is coupled to the stimulatory Gs-protein, and receptor stimulation results in activation of adenylyl cyclase (AC leading to a rise of cAMP concentration. In addition, this receptor is coupled to the G12-protein to activate small GTPases of the Rho family. This review focuses on molecular mechanisms responsible for the 5-HT7 receptor-mediated signaling. We provide detailed overview of signaling cascades controlled and regulated by the 5-HT7 receptor and discuss the functional impact of 5-HT7 receptor for the regulation of different cellular and subcellular processes.

  2. 5-HT7 receptor-mediated fear conditioning and possible involvement of extracellular signal-regulated kinase.

    Science.gov (United States)

    Takeda, Kotaro; Tsuji, Minoru; Miyagawa, Kazuya; Takeda, Hiroshi

    2017-01-18

    Fear conditioning is a valuable behavioral paradigm for studying the neural basis of emotional learning and memory. The present study examined the involvement of extracellular signal-regulated kinase 1/2 (ERK) signaling on the serotonin (5-HT)7 receptor-mediated fear conditioning. Conditioning was performed in a trial in which a tone was followed by an electrical foot-shock. Context- and tone-dependent fear were examined in tests conducted 24 and 48h after conditioning, respectively. The selective 5-HT7 receptor antagonist 2a-[4-(4-phenyl-1,2,3,6-tetrahydropyridyl)butyl]-2a,3,4,-tetrahydrobenzo(c,d)indol-2-(1H)-one (DR4004) (5mg/kg), when administered intraperitoneally (i.p.) immediately after conditioning, caused a significant decrease in both context- and tone-dependent fear responses (freezing behavior). A significant increase in ERK activity was observed in the amygdala of mice that displayed context- or tone-dependent fear responses, and these changes were also inhibited by the administration of DR4004 (5mg/kg, i.p.) immediately after conditioning. In contrast, the increase in hippocampal ERK activity in mice that displayed context-dependent fear responses was further enhanced by the administration of DR4004 (5mg/kg, i.p.). These results suggest that 5-HT7 receptor-mediated ERK signaling may play a significant role in the processes of emotional learning and memory.

  3. Receptor-Mediated Surface Charge Inversion Platform Based on Porous Silicon Nanoparticles for Efficient Cancer Cell Recognition and Combination Therapy.

    Science.gov (United States)

    Zhang, Feng; Correia, Alexandra; Mäkilä, Ermei; Li, Wei; Salonen, Jarno; Hirvonen, Jouni J; Zhang, Hongbo; Santos, Hélder A

    2017-03-22

    Negatively charged surface-modified drug delivery systems are promising for in vivo applications as they have more tendency to accumulate in tumor tissues. However, the inefficient cell uptake of these systems restricts their final therapeutic performance. Here, we have fabricated a receptor-mediated surface charge inversion nanoparticle made of undecylenic acid modified, thermally hydrocarbonized porous silicon (UnTHCPSi) nanoparticles core and sequentially modified with polyethylenimine (PEI), methotrexate (MTX), and DNA aptamer AS1411 (herein termed as UnTHCPSi-PEI-MTX@AS1411) for enhancing the cell uptake of nucleolin-positive cells. The efficient interaction of AS1411 and the relevant receptor nucleolin caused the disintegration of the negative-charged AS1411 surface. The subsequent surface charge inversion and exposure of the active targeting ligand, MTX, enhanced the cell uptake of the nanoparticles. On the basis of this synergistic effect, the UnTHCPSi-PEI-MTX@AS1411 (hydrodynamic diameter is 242 nm) were efficiently internalized by nucleolin-positive MDA-MB-231 breast cancer cells, with an efficiency around 5.8 times higher than that of nucleolin-negative cells (NIH 3T3 fibroblasts). The receptor competition assay demonstrated that the major mechanism (more than one-half) of the internalized nanoparticles in MDA-MB-231 cells was due to the receptor-mediated surface charge inversion process. Finally, after loading of sorafenib, the nanosystem showed efficient performance for combination therapy with an inhibition ratio of 35.6%.

  4. Sucrose-induced analgesia in mice: Role of nitric oxide and opioid receptor-mediated system

    Directory of Open Access Journals (Sweden)

    Abtin Shahlaee

    2013-01-01

    Full Text Available Background: The mechanism of action of sweet substance-induced analgesia is thought to involve activation of the endogenous opioid system. The nitric oxide (NO pathway has a pivotal role in pain modulation of analgesic compounds such as opioids. Objectives: We investigated the role of NO and the opioid receptor-mediated system in the analgesic effect of sucrose ingestion in mice. Materials and Methods: We evaluated the effect of intraperitoneal administration of 10 mg/kg of NO synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME and 20 mg/kg of opioid receptor antagonist, naltrexone on the tail flick response in sucrose ingesting mice. Results: Sucrose ingestion for 12 days induced a statistically significant increase in the latency of tail flick response which was unmodified by L-NAME, but partially inhibited by naltrexone administration. Conclusions: Sucrose-induced nociception may be explained by facilitating the release of endogenous opioid peptides. Contrary to some previously studied pain models, the NO/cyclic guanosine monophosphate (cGMP pathway had no role in thermal hyperalgesia in our study. We recommend further studies on the involvement of NO in other animals and pain models.

  5. PGE2 Modulates GABAA Receptors via an EP1 Receptor-Mediated Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Guang Yang

    2015-07-01

    Full Text Available Aims: PGE2 is one of the most abundant prostanoids in mammalian tissues, but its effect on neuronal receptors has not been well investigated. This study examines the effect of PGE2 on GABAA receptor currents in rat cerebellar granule neurons. Methods: GABAA currents were recorded using a patch-clamp technique. Cell surface and total protein of GABAA β1/2/3 subunits was carried out by Western blot analysis. Results: Upon incubation of neurons with PGE2 (1 µM for 60 minutes, GABAA currents were significantly potentiated. This PGE2-driven effect could be blocked by PKC or CaMKII inhibitors as well as EP1 receptor antagonist, and mimicked by PMA or EP1 receptor agonist. Furthermore, Western blot data showed that PGE2 did not increase the total expression level of GABAA receptors, but significantly increased surface levels of GABAA β1/2/3 subunits after 1 h of treatment. Consistently, both PKC and CaMKII inhibitors were able to reduce PGE2-induced increases in cell surface expression of GABAA receptors. Conclusion: Activation of either the PKC or CaMKII pathways by EP1 receptors mediates the PGE2-induced increase in GABAA currents. This suggests that upregulation of postsynaptic GABAA receptors by PGE2 may have profound effects on cerebellar functioning under physiological and pathological conditions.

  6. Prostaglandin E2 potentiation of P2X3 receptor mediated currents in dorsal root ganglion neurons

    Directory of Open Access Journals (Sweden)

    Huang Li-Yen

    2007-08-01

    Full Text Available Abstract Prostaglandin E2 (PGE2 is a well-known inflammatory mediator that enhances the excitability of DRG neurons. Homomeric P2X3 and heteromeric P2X2/3 receptors are abundantly expressed in dorsal root ganglia (DRG neurons and participate in the transmission of nociceptive signals. The interaction between PGE2 and P2X3 receptors has not been well delineated. We studied the actions of PGE2 on ATP-activated currents in dissociated DRG neurons under voltage-clamp conditions. PGE2 had no effects on P2X2/3 receptor-mediated responses, but significantly potentiated fast-inactivating ATP currents mediated by homomeric P2X3 receptors. PGE2 exerted its action by activating EP3 receptors. To study the mechanism underlying the action of PGE2, we found that the adenylyl cyclase activator, forskolin and the membrane-permeable cAMP analogue, 8-Br-cAMP increased ATP currents, mimicking the effect of PGE2. In addition, forskolin occluded the enhancement produced by PGE2. The protein kinase A (PKA inhibitors, H89 and PKA-I blocked the PGE2 effect. In contrast, the PKC inhibitor, bisindolymaleimide (Bis did not change the potentiating action of PGE2. We further showed that PGE2 enhanced α,β-meATP-induced allodynia and hyperalgesia and the enhancement was blocked by H89. These observations suggest that PGE2 binds to EP3 receptors, resulting in the activation of cAMP/PKA signaling pathway and leading to an enhancement of P2X3 homomeric receptor-mediated ATP responses in DRG neurons.

  7. DMPD: Modulation of Toll-interleukin 1 receptor mediated signaling. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15662540 Modulation of Toll-interleukin 1 receptor mediated signaling. Li X, Qin J....iated signaling. PubmedID 15662540 Title Modulation of Toll-interleukin 1 receptor media... J Mol Med. 2005 Apr;83(4):258-66. Epub 2005 Jan 21. (.png) (.svg) (.html) (.csml) Show Modulation of Toll-interleukin 1 receptor med

  8. Self-Assembly into Nanoparticles Is Essential for Receptor Mediated Uptake of Therapeutic Antisense Oligonucleotides.

    Science.gov (United States)

    Ezzat, Kariem; Aoki, Yoshitsugu; Koo, Taeyoung; McClorey, Graham; Benner, Leif; Coenen-Stass, Anna; O'Donovan, Liz; Lehto, Taavi; Garcia-Guerra, Antonio; Nordin, Joel; Saleh, Amer F; Behlke, Mark; Morris, John; Goyenvalle, Aurelie; Dugovic, Branislav; Leumann, Christian; Gordon, Siamon; Gait, Michael J; El-Andaloussi, Samir; Wood, Matthew J A

    2015-07-08

    Antisense oligonucleotides (ASOs) have the potential to revolutionize medicine due to their ability to manipulate gene function for therapeutic purposes. ASOs are chemically modified and/or incorporated within nanoparticles to enhance their stability and cellular uptake, however, a major challenge is the poor understanding of their uptake mechanisms, which would facilitate improved ASO designs with enhanced activity and reduced toxicity. Here, we study the uptake mechanism of three therapeutically relevant ASOs (peptide-conjugated phosphorodiamidate morpholino (PPMO), 2'Omethyl phosphorothioate (2'OMe), and phosphorothioated tricyclo DNA (tcDNA) that have been optimized to induce exon skipping in models of Duchenne muscular dystrophy (DMD). We show that PPMO and tcDNA have high propensity to spontaneously self-assemble into nanoparticles. PPMO forms micelles of defined size and their net charge (zeta potential) is dependent on the medium and concentration. In biomimetic conditions and at low concentrations, PPMO obtains net negative charge and its uptake is mediated by class A scavenger receptor subtypes (SCARAs) as shown by competitive inhibition and RNAi silencing experiments in vitro. In vivo, the activity of PPMO was significantly decreased in SCARA1 knockout mice compared to wild-type animals. Additionally, we show that SCARA1 is involved in the uptake of tcDNA and 2'OMe as shown by competitive inhibition and colocalization experiments. Surface plasmon resonance binding analysis to SCARA1 demonstrated that PPMO and tcDNA have higher binding profiles to the receptor compared to 2'OMe. These results demonstrate receptor-mediated uptake for a range of therapeutic ASO chemistries, a mechanism that is dependent on their self-assembly into nanoparticles.

  9. Mechanism-Based Tumor-Targeting Drug Delivery System. Validation of Efficient Vitamin Receptor-Mediated Endocytosis and Drug Release

    Energy Technology Data Exchange (ETDEWEB)

    Chen, S.; Wong, S.; Zhao, X.; Chen, J.; Chen, J.; Kuznetsova, L.; Ojima, I.

    2010-05-01

    An efficient mechanism-based tumor-targeting drug delivery system, based on tumor-specific vitamin-receptor mediated endocytosis, has been developed. The tumor-targeting drug delivery system is a conjugate of a tumor-targeting molecule (biotin: vitamin H or vitamin B-7), a mechanism-based self-immolative linker and a second-generation taxoid (SB-T-1214) as the cytotoxic agent. This conjugate (1) is designed to be (i) specific to the vitamin receptors overexpressed on tumor cell surface and (ii) internalized efficiently through receptor-mediated endocytosis, followed by smooth drug release via glutathione-triggered self-immolation of the linker. In order to monitor and validate the sequence of events hypothesized, i.e., receptor-mediated endocytosis of the conjugate, drug release, and drug-binding to the target protein (microtubules), three fluorescent/fluorogenic molecular probes (2, 3, and 4) were designed and synthesized. The actual occurrence of these processes was unambiguously confirmed by means of confocal fluorescence microscopy (CFM) and flow cytometry using L1210FR leukemia cells, overexpressing biotin receptors. The molecular probe 4, bearing the taxoid linked to fluorescein, was also used to examine the cell specificity (i.e., efficacy of receptor-based cell targeting) for three cell lines, L1210FR (biotin receptors overexpressed), L1210 (biotin receptors not overexpressed), and WI38 (normal human lung fibroblast, biotin receptor negative). As anticipated, the molecular probe 4 exhibited high specificity only to L1210FR. To confirm the direct correlation between the cell-specific drug delivery and anticancer activity of the probe 4, its cytotoxicity against these three cell lines was also examined. The results clearly showed a good correlation between the two methods. In the same manner, excellent cell-specific cytotoxicity of the conjugate 1 (without fluorescein attachment to the taxoid) against the same three cell lines was confirmed. This mechanism

  10. P2X receptor-mediated ATP purinergic signaling in health and disease

    Directory of Open Access Journals (Sweden)

    Jiang LH

    2012-09-01

    Full Text Available Lin-Hua JiangSchool of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, United KingdomAbstract: Purinergic P2X receptors are plasma membrane proteins present in a wide range of mammalian cells where they act as a cellular sensor, enabling cells to detect and respond to extracellular adenosine triphosphate (ATP, an important signaling molecule. P2X receptors function as ligand-gated Ca2+-permeable cationic channels that open upon ATP binding to elevate intracellular Ca2+ concentrations and cause membrane depolarization. In response to sustained activation, P2X receptors induce formation of a pore permeable to large molecules. P2X receptors also interact with distinct functional proteins and membrane lipids to form specialized signaling complexes. Studies have provided compelling evidence to show that such P2X receptor-mediated ATP-signaling mechanisms determine and regulate a growing number and diversity of important physiological processes, including neurotransmission, muscle contraction, and cytokine release. There is accumulating evidence to support strong causative relationships of altered receptor expression and function with chronic pain, inflammatory diseases, cancers, and other pathologies or diseases. Numerous high throughput screening drug discovery programs and preclinical studies have thus far demonstrated the proof of concepts that the P2X receptors are druggable targets and selective receptor antagonism is a promising therapeutics approach. This review will discuss the recent progress in understanding the mammalian P2X receptors with respect to the ATP-signaling mechanisms, physiological and pathophysiological roles, and development and preclinical studies of receptor antagonists.Keywords: extracellular ATP, ion channel, large pore, signaling complex, chronic pain, inflammatory diseases

  11. Brain delta2 opioid receptors mediate SNC-80-evoked hypothermia in rats.

    Science.gov (United States)

    Rawls, Scott Manning; Hewson, Jennifer Marie; Inan, Saadet; Cowan, Alan

    2005-07-05

    Despite insights into an increasingly significant role for delta opioid receptors in thermoregulation, it is unclear whether delta receptors located in the brain or periphery play the more critical role in body temperature regulation. Moreover, it is not entirely clear which delta receptor phenotype, delta1 or delta2, mediates the hypothermic actions of delta agonists. Because SNC-80 distributes into central and peripheral compartments and produces rapid hypothermia following systemic injection, the nonpeptide delta agonist is particularly useful in discriminating the site of action of delta receptor-mediated hypothermia. To determine the locus and phenotype of delta receptor which mediates SNC-80-induced hypothermia, we injected SNC-80 and phenotype selective delta antagonists to male Sprague-Dawley rats. SNC-80 (10-50 mg/kg, im) evoked hypothermia that peaked 30 min post-injection. Naltrexone (5 mg/kg, sc), an opioid antagonist, or naltrindole (5 mg/kg, sc), a delta antagonist, blocked the hypothermic response to SNC-80 (35 mg/kg, im). The hypothermia caused by SNC-80 (35 mg/kg, im) was blocked by a delta2 antagonist, naltriben (2.5 mg/kg, sc), but was not affected by BNTX (5 and 10 mg/kg, sc), a delta1 antagonist. The administration of naltriben (10 microg/rat, icv) 30 min before SNC-80 (35 mg/kg, im) prevented SNC-80-evoked hypothermia. In contrast, methylnaltrexone (5 mg/kg, sc), a peripherally restricted opioid antagonist, did not affect the hypothermia caused by SNC-80. The present data demonstrate that selective activation of brain delta2 receptors is a major mechanism of SNC-80-evoked hypothermia in rats.

  12. P2 receptor-mediated signaling in mast cell biology

    OpenAIRE

    Bulanova, Elena; Bulfone-Paus, Silvia

    2009-01-01

    Mast cells are widely recognized as effector cells of allergic inflammatory reactions. They contribute to the pathogenesis of different chronic inflammatory diseases, wound healing, fibrosis, thrombosis/fibrinolysis, and anti-tumor immune responses. In this paper, we summarized the role of P2X and P2Y receptors in mast cell activation and effector functions. Mast cells are an abundant source of ATP which is stored in their granules and secreted upon activation. We discuss the contribution of ...

  13. NFAT regulates calcium-sensing receptor-mediated TNF production

    Energy Technology Data Exchange (ETDEWEB)

    abdullah, huda ismail; Pedraza, Paulina L.; Hao, Shoujin; Rodland, Karin D.; McGiff, John C.; Ferreri, Nicholas R.

    2006-05-01

    Because nuclear factor of activated T cells (NFAT) has been implicated in TNF production as well as osmoregulation and salt and water homeostasis, we addressed whether calcium-sensing receptor (CaR)-mediated TNF production in medullary thick ascending limb (mTAL) cells was NFAT dependent. TNF production in response to addition of extracellular Ca2+ (1.2 mM) was abolished in mTAL cells transiently transfected with a dominant-negative CaR construct (R796W) or pretreated with the phosphatidylinositol phospholipase C (PI-PLC) inhibitor U-73122. Cyclosporine A (CsA), an inhibitor of the serine/threonine phosphatase calcineurin, and a peptide ligand, VIVIT, that selectively inhibits calcineurin-NFAT signaling, also prevented CaR-mediated TNF production. Increases in calcineurin activity in cells challenged with Ca2+ were inhibited after pretreatment with U-73122 and CsA, suggesting that CaR activation increases calcineurin activity in a PI-PLC-dependent manner. Moreover, U-73122, CsA, and VIVIT inhibited CaR-dependent activity of an NFAT construct that drives expression of firefly luciferase in transiently transfected mTAL cells. Collectively, these data verify the role of calcineurin and NFAT in CaR-mediated TNF production by mTAL cells. Activation of the CaR also increased the binding of NFAT to a consensus oligonucleotide, an effect that was blocked by U-73122 and CsA, suggesting that a calcineurin- and NFAT-dependent pathway increases TNF production in mTAL cells. This mechanism likely regulates TNF gene transcription as U-73122, CsA, and VIVIT blocked CaR-dependent activity of a TNF promoter construct. Elucidating CaR-mediated signaling pathways that regulate TNF production in the mTAL will be crucial to understanding mechanisms that regulate extracellular fluid volume and salt balance.

  14. NFAT regulates calcium-sensing receptor-mediated TNF production.

    Science.gov (United States)

    Abdullah, Huda Ismail; Pedraza, Paulina L; Hao, Shoujin; Rodland, Karin D; McGiff, John C; Ferreri, Nicholas R

    2006-05-01

    Because nuclear factor of activated T cells (NFAT) has been implicated in TNF production as well as osmoregulation and salt and water homeostasis, we addressed whether calcium-sensing receptor (CaR)-mediated TNF production in medullary thick ascending limb (mTAL) cells was NFAT dependent. TNF production in response to addition of extracellular Ca(2+) (1.2 mM) was abolished in mTAL cells transiently transfected with a dominant-negative CaR construct (R796W) or pretreated with the phosphatidylinositol phospholipase C (PI-PLC) inhibitor U-73122. Cyclosporine A (CsA), an inhibitor of the serine/threonine phosphatase calcineurin, and a peptide ligand, VIVIT, that selectively inhibits calcineurin-NFAT signaling, also prevented CaR-mediated TNF production. Increases in calcineurin activity in cells challenged with Ca(2+) were inhibited after pretreatment with U-73122 and CsA, suggesting that CaR activation increases calcineurin activity in a PI-PLC-dependent manner. Moreover, U-73122, CsA, and VIVIT inhibited CaR-dependent activity of an NFAT construct that drives expression of firefly luciferase in transiently transfected mTAL cells. Collectively, these data verify the role of calcineurin and NFAT in CaR-mediated TNF production by mTAL cells. Activation of the CaR also increased the binding of NFAT to a consensus oligonucleotide, an effect that was blocked by U-73122 and CsA, suggesting that a calcineurin- and NFAT-dependent pathway increases TNF production in mTAL cells. This mechanism likely regulates TNF gene transcription as U-73122, CsA, and VIVIT blocked CaR-dependent activity of a TNF promoter construct. Elucidating CaR-mediated signaling pathways that regulate TNF production in the mTAL will be crucial to understanding mechanisms that regulate extracellular fluid volume and salt balance.

  15. Receptor-mediated Ca2+ and PKC signaling triggers the loss of cortical PKA compartmentalization through the redistribution of gravin.

    Science.gov (United States)

    Schott, Micah B; Grove, Bryon

    2013-11-01

    A-Kinase Anchoring Proteins (AKAPs) direct the flow of cellular information by positioning multiprotein signaling complexes into proximity with effector proteins. However, certain AKAPs are not stationary but can undergo spatiotemporal redistribution in response to stimuli. Gravin, a 300kD AKAP that intersects with a diverse signaling array, is localized to the plasma membrane but has been shown to translocate to the cytosol following the elevation of intracellular calcium ([Ca(2+)]i). Despite the potential for gravin redistribution to impact multiple signaling pathways, the dynamics of this event remain poorly understood. In this study, quantitative microscopy of cells expressing gravin-EGFP revealed that Ca(2+) elevation caused the complete translocation of gravin from the cell cortex to the cytosol in as little as 60s of treatment with ionomycin or thapsigargin. In addition, receptor mediated signaling was also shown to cause gravin redistribution following ATP treatment, and this event required both [Ca(2+)]i elevation and PKC activation. To understand the mechanism for Ca(2+) mediated gravin dynamics, deletion of calmodulin-binding domains revealed that a fourth putative calmodulin binding domain called CB4 (a.a. 670-694) is critical for targeting gravin to the cell cortex despite its location downstream of gravin's membrane-targeting domains, which include an N-terminal myristoylation site and three polybasic domains. Finally, confocal microscopy of cells co-transfected with gravin-EYFP and PKA RII-ECFP revealed that gravin redistribution mediated by ionomycin, thapsigargin, and ATP each triggered the gravin-dependent loss of PKA localized at the cell cortex. Our results support the hypothesis that gravin redistribution regulates cross-talk between PKA-dependent signaling and receptor-mediated events involving Ca(2+) and PKC. © 2013.

  16. Intracellular Ca2+ release through ryanodine receptors contributes to AMPA receptor-mediated mitochondrial dysfunction and ER stress in oligodendrocytes

    Science.gov (United States)

    Ruiz, A; Matute, C; Alberdi, E

    2010-01-01

    Overactivation of ionotropic glutamate receptors in oligodendrocytes induces cytosolic Ca2+ overload and excitotoxic death, a process that contributes to demyelination and multiple sclerosis. Excitotoxic insults cause well-characterized mitochondrial alterations and endoplasmic reticulum (ER) dysfunction, which is not fully understood. In this study, we analyzed the contribution of ER-Ca2+ release through ryanodine receptors (RyRs) and inositol triphosphate receptors (IP3Rs) to excitotoxicity in oligodendrocytes in vitro. First, we observed that oligodendrocytes express all previously characterized RyRs and IP3Rs. Blockade of Ca2+-induced Ca2+ release by TMB-8 following α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated insults attenuated both oligodendrocyte death and cytosolic Ca2+ overload. In turn, RyR inhibition by ryanodine reduced as well the Ca2+ overload whereas IP3R inhibition was ineffective. Furthermore, AMPA-triggered mitochondrial membrane depolarization, oxidative stress and activation of caspase-3, which in all instances was diminished by RyR inhibition. In addition, we observed that AMPA induced an ER stress response as revealed by α subunit of the eukaryotic initiation factor 2α phosphorylation, overexpression of GRP chaperones and RyR-dependent cleavage of caspase-12. Finally, attenuating ER stress with salubrinal protected oligodendrocytes from AMPA excitotoxicity. Together, these results show that Ca2+ release through RyRs contributes to cytosolic Ca2+ overload, mitochondrial dysfunction, ER stress and cell death following AMPA receptor-mediated excitotoxicity in oligodendrocytes. PMID:21364659

  17. Nuclear receptors : mediators and modifiers of inflammation-induced cholestasis

    NARCIS (Netherlands)

    Mulder, Jaap; Karpen, Saul J.; Tietge, Uwe J. F.; Kuipers, Folkert

    2009-01-01

    Inflammation-induced cholestasis (IIC) is a frequently occurring phenomenon. A central role in its pathogenesis is played by nuclear receptors (NRs). These ligand-activated transcription factors not only regulate basal expression of hepatobiliary transport systems, but also mediate adaptive response

  18. P2 receptor-mediated signaling in mast cell biology.

    Science.gov (United States)

    Bulanova, Elena; Bulfone-Paus, Silvia

    2010-03-01

    Mast cells are widely recognized as effector cells of allergic inflammatory reactions. They contribute to the pathogenesis of different chronic inflammatory diseases, wound healing, fibrosis, thrombosis/fibrinolysis, and anti-tumor immune responses. In this paper, we summarized the role of P2X and P2Y receptors in mast cell activation and effector functions. Mast cells are an abundant source of ATP which is stored in their granules and secreted upon activation. We discuss the contribution of mast cells to the extracellular ATP release and to the maintenance of extracellular nucleotides pool. Recent publications highlight the importance of purinergic signaling for the pathogenesis of chronic airway inflammation. Therefore, the role of ATP and P2 receptors in allergic inflammation with focus on mast cells was analyzed. Finally, ATP functions as mast cell autocrine/paracrine factor and as messenger in intercellular communication between mast cells, nerves, and glia in the central nervous system.

  19. Neurotransmitter receptor-mediated signaling pathways as modulators of carcinogenesis.

    Science.gov (United States)

    Schuller, Hildegard M

    2007-01-01

    The autonomic nervous system with its two antagonistic branches, the sympathicus and the parasympathicus, regulates the activities of all body functions that are not under voluntary control. While the autonomic regulation of organ functions has been extensively studied, little attention has been given to the potential role of neurohumoral transmission at the cellular level in the development of cancer. Studies conducted by our laboratory first showed that binding of the parasympathetic neurotransmitter, acetylcholine, as well as nicotine or its nitrosated cancer-causing derivative, NNK, to nicotinic acetylcholine receptors comprised of alpha7 subunits activated a mitogenic signal transduction pathway in normal and neoplastic pulmonary neuroendocrine cells. On the other hand, beta-adrenergic receptors (Beta-ARs), which transmit signals initiated by binding of the catecholamine neurotransmitters of the sympathicus, were identified by our laboratory as important regulators of cell proliferation in cell lines derived from human adenocarcinomas of the lungs, pancreas, and breast. The tobacco-specific carcinogen NNK bound with high affinity to Beta1- and Beta2-ARs, thus activating cAMP, protein kinase A, and the transcription factor CREB. Collectively, neurotransmitter receptors of the nicotinic and Beta-adrenergic families appear to regulate cellular functions essential for the development and survival of the most common human cancers.

  20. Vascular endothelin ET(B) receptor-mediated contraction requires phosphorylation of ERK1/2 proteins

    DEFF Research Database (Denmark)

    Luo, Guogang; Jamali, Roya; Cao, Yong-Xiao;

    2006-01-01

    RNA and protein expressions. The endothelin ET(B) receptor-mediated contraction was associated with increase in phosphorylation of extracellular regulation kinase 1 and 2 (ERK1/2) proteins and elevated levels of intracellular calcium. The elevation curve of intracellular calcium consisted of two phases: one rapid...... and one sustained. Inhibition of ERK1/2 phosphorylation by SB386023 or blockage of calcium channels by nifedipine significantly reduced the endothelin ET(B) receptor-mediated contraction (P..., phosphorylation of ERK1/2 proteins and elevation of intracellular calcium level are required for endothelin ET(B) receptor-mediated contraction in rat mesenteric artery....

  1. Upregulation of endothelin ETB receptor-mediated vasoconstriction in rat coronary artery after organ culture

    DEFF Research Database (Denmark)

    Eskesen, Karen; Edvinsson, Lars

    2006-01-01

    The aim of this study was to examine if endothelin ET(B) receptor-mediated contraction occurred in isolated segments of rat coronary arteries during organ culture. Presence of contractile endothelin ET(B) receptors was studied by measuring the change in isometric tension in rings of left anterior...... descending coronary arteries isolated from hearts of rats as response to application of the selective endothelin ET(B) receptor agonist, Sarafotoxin 6c and endothelin-1. In segments cultured 1 day in serum free Dulbecco's Modified Eagle's Medium, Sarafotoxin 6c induced a concentration dependent contraction......(+)-solution was not modified after 1 day in culture medium. The experiments indicate that organ culture of rat coronary arteries upregulate endothelin ET(B) receptor-mediated contraction by inducing synthesis of new protein....

  2. Receptor-mediated choreography of life and death.

    Science.gov (United States)

    Bhardwaj, Anjana; Aggarwal, Bharat B

    2003-09-01

    The cytokine tumor necrosis factor was originally identified as a protein that kills tumor cells. So far, 18 distinct members of this family have been identified. All of them regulate cell survival, proliferation, differentiation, and cell death, also called apoptosis. The apoptosis induced by TNF, and other members of the family, for example, FasL, VEGI, and TRAIL is mediated through death receptors. The apoptotic signals by these cytokines are transduced by eight different death domain- (DD) containing receptors (TNFR1, also called DR1; Fas, also called DR2; DR3, DR4, DR5, DR6, NGFR, and EDAR). The intracellular portion of all these receptors contains a region approximately 80 amino acids long referred to as the "death domain." Upon activation by its ligand, the DD recruits various proteins that mediate both death and proliferation of the cells. These proteins in turn recruit other proteins via their DDs or death effector domains. The actual destruction of the cell, however, is accomplished by serial activation of a family of proteases referred to as caspases. Cell death is negatively regulated by a family of proteins that includes decoy receptors, silencer of DD, sentrin, cellular FLICE inhibitory protein, cellular inhibitors of apoptosis, and survivin. This review is an attempt to describe how these negative and positive players of cell death perform a harmonious dance with each other.

  3. Experimental Cannabinoid 2 Receptor-Mediated Immune Modulation in Sepsis

    Directory of Open Access Journals (Sweden)

    J. Sardinha

    2014-01-01

    Full Text Available Sepsis is a complex condition that results from a dysregulated immune system in response to a systemic infection. Current treatments lack effectiveness in reducing the incidence and mortality associated with this disease. The endocannabinoid system offers great promise in managing sepsis pathogenesis due to its unique characteristics. The present study explored the effect of modulating the CB2 receptor pathway in an acute sepsis mouse model. Endotoxemia was induced by intravenous injection of lipopolysaccharide (LPS in mice and intestinal microcirculation was assessed through intravital microscopy. We found that HU308 (CB2 receptor agonist reduced the number of adherent leukocytes in submucosal venules but did not restore muscular and mucosal villi FCD in endotoxemic mice. AM630 (CB2 receptor antagonist maintained the level of adherent leukocytes induced by LPS but further reduced muscular and mucosal villi FCD. URB597 (FAAH inhibitor and JZL184 (MAGL inhibitor both reduced the number of adherent leukocytes in submucosal venules but did not restore the mucosal villi FCD. Using various compounds we have shown different mechanisms of activating CB2 receptors to reduce leukocyte endothelial interactions in order to prevent further inflammatory damage during sepsis.

  4. P2X7 receptors mediate ischemic damage to oligodendrocytes.

    Science.gov (United States)

    Domercq, Maria; Perez-Samartin, Alberto; Aparicio, David; Alberdi, Elena; Pampliega, Olatz; Matute, Carlos

    2010-04-15

    Brain ischemia leading to stroke is a major cause of disability in developed countries. Therapeutic strategies have most commonly focused on protecting neurons from ischemic damage. However, ischemic damage to white matter causes oligodendrocyte death, myelin disruption, and axon dysfunction, and it is partially mediated by glutamate excitotoxicity. We have previously demonstrated that oligodendrocytes express ionotropic purinergic receptors. The objective of this study was to investigate the role of purinergic signaling in white matter ischemia. We show that, in addition to glutamate, enhanced ATP signaling during ischemia is also deleterious to oligodendrocytes and myelin, and impairs white matter function. Thus, ischemic oligodendrocytes in culture display an inward current and cytosolic Ca(2+) overload, which is partially mediated by P2X7 receptors. Indeed, oligodendrocytes release ATP after oxygen and glucose deprivation through the opening of pannexin hemichannels. Consistently, ischemia-induced mitochondrial depolarization as well as oxidative stress culminating in cell death are partially reversed by P2X7 receptor antagonists, by the ATP degrading enzyme apyrase and by blockers of pannexin hemichannels. In turn, ischemic damage in isolated optic nerves, which share the properties of brain white matter, is greatly attenuated by all these drugs. Ultrastructural analysis and electrophysiological recordings demonstrated that P2X7 antagonists prevent ischemic damage to oligodendrocytes and myelin, and improved action potential recovery after ischemia. These data indicate that ATP released during ischemia and the subsequent activation of P2X7 receptor is critical to white matter demise during stroke and point to this receptor type as a therapeutic target to limit tissue damage in cerebrovascular diseases.

  5. Ligand Receptor-Mediated Regulation of Growth in Plants.

    Science.gov (United States)

    Haruta, Miyoshi; Sussman, Michael R

    2017-01-01

    Growth and development of multicellular organisms are coordinately regulated by various signaling pathways involving the communication of inter- and intracellular components. To form the appropriate body patterns, cellular growth and development are modulated by either stimulating or inhibiting these pathways. Hormones and second messengers help to mediate the initiation and/or interaction of the various signaling pathways in all complex multicellular eukaryotes. In plants, hormones include small organic molecules, as well as larger peptides and small proteins, which, as in animals, act as ligands and interact with receptor proteins to trigger rapid biochemical changes and induce the intracellular transcriptional and long-term physiological responses. During the past two decades, the availability of genetic and genomic resources in the model plant species, Arabidopsis thaliana, has greatly helped in the discovery of plant hormone receptors and the components of signal transduction pathways and mechanisms used by these immobile but highly complex organisms. Recently, it has been shown that two of the most important plant hormones, auxin and abscisic acid (ABA), act through signaling pathways that have not yet been recognized in animals. For example, auxins stimulate cell elongation by bringing negatively acting transcriptional repressor proteins to the proteasome to be degraded, thus unleashing the gene expression program required for increasing cell size. The "dormancy" inducing hormone, ABA, binds to soluble receptor proteins and inhibits a specific class of protein phosphatases (PP2C), which activates phosphorylation signaling leading to transcriptional changes needed for the desiccation of the seeds prior to entering dormancy. While these two hormone receptors have no known animal counterparts, there are also many similarities between animal and plant signaling pathways. For example, in plants, the largest single gene family in the genome is the protein kinase

  6. Inhibitory effects of benzodiazepines on the adenosine A(2B) receptor mediated secretion of interleukin-8 in human mast cells.

    Science.gov (United States)

    Hoffmann, Kristina; Xifró, Rosa Altarcheh; Hartweg, Julia Lisa; Spitzlei, Petra; Meis, Kirsten; Molderings, Gerhard J; von Kügelgen, Ivar

    2013-01-30

    The activation of adenosine A(2B) receptors in human mast cells causes pro-inflammatory responses such as the secretion of interleukin-8. There is evidence for an inhibitory effect of benzodiazepines on mast cell mediated symptoms in patients with systemic mast cell activation disease. Therefore, we investigated the effects of benzodiazepines on adenosine A(2B) receptor mediated interleukin-8 production in human mast cell leukaemia (HMC1) cells by an enzyme linked immunosorbent assay. The adenosine analogue N-ethylcarboxamidoadenosine (NECA, 0.3-3 μM) increased interleukin-8 production about 5-fold above baseline. This effect was attenuated by the adenosine A(2B) receptor antagonist MRS1754 (N-(4-cyanophenyl)-2-{4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy}-acetamide) 1 μM. In addition, diazepam, 4'-chlorodiazepam and flunitrazepam (1-30 μM) markedly reduced NECA-induced interleukin-8 production in that order of potency, whereas clonazepam showed only a modest inhibition. The inhibitory effect of diazepam was not altered by flumazenil 10 μM or PK11195 (1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide) 10 μM. Diazepam attenuated the NECA-induced expression of mRNA encoding for interleukin-8. Moreover, diazepam and flunitrazepam reduced the increasing effects of NECA on cAMP-response element- and nuclear factor of activated t-cells-driven luciferase reporter gene activities in HMC1 cells. Neither diazepam nor flunitrazepam affected NECA-induced increases in cellular cAMP levels in CHO Flp-In cells stably expressing recombinant human adenosine A(2B) receptors, excluding a direct action of benzodiazepines on human adenosine A(2B) receptors. In conclusion, this is the first study showing an inhibitory action of benzodiazepines on adenosine A(2B) receptor mediated interleukin-8 production in human mast (HMC1) cells. The rank order of potency indicates the involvement of an atypical benzodiazepine binding site.

  7. Mu-Opioid (MOP) receptor mediated G-protein signaling is impaired in specific brain regions in a rat model of schizophrenia.

    Science.gov (United States)

    Szűcs, Edina; Büki, Alexandra; Kékesi, Gabriella; Horváth, Gyöngyi; Benyhe, Sándor

    2016-04-21

    Schizophrenia is a complex mental health disorder. Clinical reports suggest that many patients with schizophrenia are less sensitive to pain than other individuals. Animal models do not interpret schizophrenia completely, but they can model a number of symptoms of the disease, including decreased pain sensitivities and increased pain thresholds of various modalities. Opioid receptors and endogenous opioid peptides have a substantial role in analgesia. In this biochemical study we investigated changes in the signaling properties of the mu-opioid (MOP) receptor in different brain regions, which are involved in the pain transmission, i.e., thalamus, olfactory bulb, prefrontal cortex and hippocampus. Our goal was to compare the transmembrane signaling mediated by MOP receptors in control rats and in a recently developed rat model of schizophrenia. Regulatory G-protein activation via MOP receptors were measured in [(35)S]GTPγS binding assays in the presence of a highly selective MOP receptor peptide agonist, DAMGO. It was found that the MOP receptor mediated activation of G-proteins was substantially lower in membranes prepared from the 'schizophrenic' model rats than in control animals. The potency of DAMGO to activate MOP receptor was also decreased in all brain regions studied. Taken together in our rat model of schizophrenia, MOP receptor mediated G-proteins have a reduced stimulatory activity compared to membrane preparations taken from control animals. The observed distinct changes of opioid receptor functions in different areas of the brain do not explain the augmented nociceptive threshold described in these animals.

  8. Histaminergic H1 receptors mediate L-histidine-induced anxiety in elevated plus-maze test in mice.

    Science.gov (United States)

    Kumar, Kuchibhotla Vijaya; Krishna, Devarakonda Rama; Palit, Gautam

    2007-05-01

    The central histaminergic system is reported to mediate behavioural, hormonal and physiological homeostasis of living organisms. Recent reports indicate its prominent role in various neurobehavioural disorders such as depression and psychosis. This study evaluated the effect of activation of the central histaminergic system in anxiety-like conditions, using the elevated plus-maze test in mice, and elucidated the role of different histaminergic receptors mediating such effects. Peripheral administration of L-histidine (L-His), in a dose-dependent manner, significantly decreased the exploration time in open arms and number of entries into open arms without modifying the number of entries into closed arms of the elevated plus-maze, indicating anxiogenesis. Further, such effects of central histamine were significantly attenuated, in a dose-dependent manner, by pretreatment with pyrilamine (H1 receptor antagonist). Pretreatment with either zolantidine (H2 receptor antagonist) or thioperamide (H3 receptor antagonist), however, failed to attenuate the L-His-induced anxiogenesis. Our results indicate that anxiogenic effects of central histaminergic system appear to be mediated prominently by activation of H1 receptors.

  9. Receptor-mediated binding and uptake of GnRH agonist and antagonist by pituitary cells

    Energy Technology Data Exchange (ETDEWEB)

    Jennes, L.; Stumpf, W.E.; Conn, P.M.

    1984-01-01

    The intracellular pathway of an enzyme resistant GnRH agonist (D- Lys6 -GnRH) conjugated to ferritin or to colloidal gold was followed in cultured pituitary cells. After an initial uniform distribution over the cell surface of gonadotropes, the electrondense marker was internalized, either individually or in small groups. After longer incubation times, the marker appeared in the lysosomal compartment and the Golgi apparatus, where it could be found in the vesicular as well as cisternal portion. In addition, the receptor-mediated endocytosis of the GnRH antagonist D-p-Glu1-D-Phe2-D-Trp3-D- Lys6 -GnRH was studied by light and electron microscopic autoradiography after 30 and 60 min of incubation to ensure uptake. At both time points, in in vitro as well as in vivo studies, silver grains were localized over cytoplasmic organelles of castration cells, including dilated endoplasmic reticulum, lysosomes, and clear vesicles. No consistent association with cell nuclei, mitochondria, or secretory vesicles could be observed. The results suggest that both agonist and antagonist are binding selectively to the plasma membrane of gonadotropes and subsequently are taken up via receptor-mediated endocytosis for degradation or possible action on synthetic processes.

  10. Regulation and ontogeny of subtypes of muscarinic receptors and muscarinic receptor-mediated

    Energy Technology Data Exchange (ETDEWEB)

    Lee, W.

    1989-01-01

    The densities of total and M1 muscarinic receptors were measured using the muscarinic receptor antagonists {sup 3}H-quinuclidinyl benzilate and {sup 3}H-pirenzepine, respectively. Thus, the difference between the density of {sup 3}H-quinuclidinyl benzilate and {sup 3}H-pirenzepine binding sites represents the density of M2 sites. In addition, there is no observable change in either acetylcholine-stimulated phosphoinositide breakdown (suggested to be an M1 receptor-mediated response) or in carbachol-mediated inhibition of cyclic AMP accumulation (suggested to be an M2 receptor-mediated response) in slices of cortex+dorsal hippocampus following chronic atropine administration. In other experiments, it has been shown that the M1 and M2 receptors in rat cortex have different ontogenetic profiles. The M2 receptor is present at adult levels at birth, while the M1 receptor develops slowly from low levels at postnatal week 1 to adult levels at postnatal week 3. The expression of acetylcholine-stimulated phosphoinositide breakdown parallels the development of M1 receptors, while the development of carbachol-mediated inhibition of cyclic AMP accumulation occurs abruptly between weeks 2 and 3 postnatally.

  11. Target shape dependence in a simple model of receptor-mediated endocytosis and phagocytosis.

    Science.gov (United States)

    Richards, David M; Endres, Robert G

    2016-05-31

    Phagocytosis and receptor-mediated endocytosis are vitally important particle uptake mechanisms in many cell types, ranging from single-cell organisms to immune cells. In both processes, engulfment by the cell depends critically on both particle shape and orientation. However, most previous theoretical work has focused only on spherical particles and hence disregards the wide-ranging particle shapes occurring in nature, such as those of bacteria. Here, by implementing a simple model in one and two dimensions, we compare and contrast receptor-mediated endocytosis and phagocytosis for a range of biologically relevant shapes, including spheres, ellipsoids, capped cylinders, and hourglasses. We find a whole range of different engulfment behaviors with some ellipsoids engulfing faster than spheres, and that phagocytosis is able to engulf a greater range of target shapes than other types of endocytosis. Further, the 2D model can explain why some nonspherical particles engulf fastest (not at all) when presented to the membrane tip-first (lying flat). Our work reveals how some bacteria may avoid being internalized simply because of their shape, and suggests shapes for optimal drug delivery.

  12. The miR-199-dynamin regulatory axis controls receptor-mediated endocytosis.

    Science.gov (United States)

    Aranda, Juan F; Canfrán-Duque, Alberto; Goedeke, Leigh; Suárez, Yajaira; Fernández-Hernando, Carlos

    2015-09-01

    Small non-coding RNAs (microRNAs) are important regulators of gene expression that modulate many physiological processes; however, their role in regulating intracellular transport remains largely unknown. Intriguingly, we found that the dynamin (DNM) genes, a GTPase family of proteins responsible for endocytosis in eukaryotic cells, encode the conserved miR-199a and miR-199b family of miRNAs within their intronic sequences. Here, we demonstrate that miR-199a and miR-199b regulate endocytic transport by controlling the expression of important mediators of endocytosis such as clathrin heavy chain (CLTC), Rab5A, low-density lipoprotein receptor (LDLR) and caveolin-1 (Cav-1). Importantly, miR-199a-5p and miR-199b-5p overexpression markedly inhibits CLTC, Rab5A, LDLR and Cav-1 expression, thus preventing receptor-mediated endocytosis in human cell lines (Huh7 and HeLa). Of note, miR-199a-5p inhibition increases target gene expression and receptor-mediated endocytosis. Taken together, our work identifies a new mechanism by which microRNAs regulate intracellular trafficking. In particular, we demonstrate that the DNM, miR-199a-5p and miR-199b-5p genes act as a bifunctional locus that regulates endocytosis, thus adding an unexpected layer of complexity in the regulation of intracellular trafficking.

  13. Adaptation in sound localization: from GABA(B) receptor-mediated synaptic modulation to perception.

    Science.gov (United States)

    Stange, Annette; Myoga, Michael H; Lingner, Andrea; Ford, Marc C; Alexandrova, Olga; Felmy, Felix; Pecka, Michael; Siveke, Ida; Grothe, Benedikt

    2013-12-01

    Across all sensory modalities, the effect of context-dependent neural adaptation can be observed at every level, from receptors to perception. Nonetheless, it has long been assumed that the processing of interaural time differences, which is the primary cue for sound localization, is nonadaptive, as its outputs are mapped directly onto a hard-wired representation of space. Here we present evidence derived from in vitro and in vivo experiments in gerbils indicating that the coincidence-detector neurons in the medial superior olive modulate their sensitivity to interaural time differences through a rapid, GABA(B) receptor-mediated feedback mechanism. We show that this mechanism provides a gain control in the form of output normalization, which influences the neuronal population code of auditory space. Furthermore, psychophysical tests showed that the paradigm used to evoke neuronal GABA(B) receptor-mediated adaptation causes the perceptual shift in sound localization in humans that was expected on the basis of our physiological results in gerbils.

  14. DMPD: Translational mini-review series on Toll-like receptors: networks regulated byToll-like receptors mediate innate and adaptive immunity. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available e receptors: networks regulated byToll-like receptors mediate innate and adaptive...ed byToll-like receptors mediate innate and adaptive immunity. Authors Parker LC, Prince LR, Sabroe I. Publi...d byToll-like receptors mediate innate and adaptive immunity. Parker LC, Prince LR, Sabroe I. Clin Exp Immun...17223959 Translational mini-review series on Toll-like receptors: networks regulate

  15. Receptor-mediated membrane adhesion of lipid-polymer hybrid (LPH) nanoparticles studied by dissipative particle dynamics simulations.

    Science.gov (United States)

    Li, Zhenlong; Gorfe, Alemayehu A

    2015-01-14

    Lipid-polymer hybrid (LPH) nanoparticles represent a novel class of targeted drug delivery platforms that combine the advantages of liposomes and biodegradable polymeric nanoparticles. However, the molecular details of the interaction between LPHs and their target cell membranes remain poorly understood. We have investigated the receptor-mediated membrane adhesion process of a ligand-tethered LPH nanoparticle using extensive dissipative particle dynamics (DPD) simulations. We found that the spontaneous adhesion process follows a first-order kinetics characterized by two distinct stages: a rapid nanoparticle-membrane engagement, followed by a slow growth in the number of ligand-receptor pairs coupled with structural re-organization of both the nanoparticle and the membrane. The number of ligand-receptor pairs increases with the dynamic segregation of ligands and receptors toward the adhesion zone causing an out-of-plane deformation of the membrane. Moreover, the fluidity of the lipid shell allows for strong nanoparticle-membrane interactions to occur even when the ligand density is low. The LPH-membrane avidity is enhanced by the increased stability of each receptor-ligand pair due to the geometric confinement and the cooperative effect arising from multiple binding events. Thus, our results reveal the unique advantages of LPH nanoparticles as active cell-targeting nanocarriers and provide some general principles governing nanoparticle-cell interactions that may aid future design of LPHs with improved affinity and specificity for a given target of interest.

  16. Receptor-mediated membrane adhesion of lipid-polymer hybrid (LPH) nanoparticles studied by dissipative particle dynamics simulations

    Science.gov (United States)

    Li, Zhenlong; Gorfe, Alemayehu A.

    2014-12-01

    Lipid-polymer hybrid (LPH) nanoparticles represent a novel class of targeted drug delivery platforms that combine the advantages of liposomes and biodegradable polymeric nanoparticles. However, the molecular details of the interaction between LPHs and their target cell membranes remain poorly understood. We have investigated the receptor-mediated membrane adhesion process of a ligand-tethered LPH nanoparticle using extensive dissipative particle dynamics (DPD) simulations. We found that the spontaneous adhesion process follows a first-order kinetics characterized by two distinct stages: a rapid nanoparticle-membrane engagement, followed by a slow growth in the number of ligand-receptor pairs coupled with structural re-organization of both the nanoparticle and the membrane. The number of ligand-receptor pairs increases with the dynamic segregation of ligands and receptors toward the adhesion zone causing an out-of-plane deformation of the membrane. Moreover, the fluidity of the lipid shell allows for strong nanoparticle-membrane interactions to occur even when the ligand density is low. The LPH-membrane avidity is enhanced by the increased stability of each receptor-ligand pair due to the geometric confinement and the cooperative effect arising from multiple binding events. Thus, our results reveal the unique advantages of LPH nanoparticles as active cell-targeting nanocarriers and provide some general principles governing nanoparticle-cell interactions that may aid future design of LPHs with improved affinity and specificity for a given target of interest.

  17. Dopaminergic enhancement of excitatory synaptic transmission in layer II entorhinal neurons is dependent on D₁-like receptor-mediated signaling.

    Science.gov (United States)

    Glovaci, I; Caruana, D A; Chapman, C A

    2014-01-31

    The modulatory neurotransmitter dopamine induces concentration-dependent changes in synaptic transmission in the entorhinal cortex, in which high concentrations of dopamine suppress evoked excitatory postsynaptic potentials (EPSPs) and lower concentrations induce an acute synaptic facilitation. Whole-cell current-clamp recordings were used to investigate the dopaminergic facilitation of synaptic responses in layer II neurons of the rat lateral entorhinal cortex. A constant bath application of 1 μM dopamine resulted in a consistent facilitation of EPSPs evoked in layer II fan cells by layer I stimulation; the size of the facilitation was more variable in pyramidal neurons, and synaptic responses in a small group of multiform neurons were not modulated by dopamine. Isolated inhibitory synaptic responses were not affected by dopamine, and the facilitation of EPSPs was not associated with a change in paired-pulse facilitation ratio. Voltage-clamp recordings of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) glutamate receptor-mediated excitatory postsynaptic currents (EPSCs) were facilitated by dopamine, but N-methyl-D-aspartate receptor-mediated currents were not. Bath application of the dopamine D₁-like receptor blocker SCH23390 (50 μM), but not the D₂-like receptor blocker sulpiride (50 μM), prevented the facilitation, indicating that it is dependent upon D₁-like receptor activation. Dopamine D₁ receptors lead to activation of protein kinase A (PKA), and including the PKA inhibitor H-89 or KT 5720 in the recording pipette solution prevented the facilitation of EPSCs. PKA-dependent phosphorylation of inhibitor 1 or the dopamine- and cAMP-regulated protein phosphatase (DARPP-32) can lead to a facilitation of AMPA receptor responses by inhibiting the activity of protein phosphatase 1 (PP1) that reduces dephosphorylation of AMPA receptors, and we found here that inhibition of PP1 occluded the facilitatory effect of dopamine. The dopamine

  18. An intracellular traffic jam: Fc receptor-mediated transport of immunoglobulin G.

    Science.gov (United States)

    Tesar, Devin B; Björkman, Pamela J

    2010-04-01

    Recent advances in imaging techniques along with more powerful in vitro and in vivo models of receptor-mediated ligand transport are facilitating advances in our understanding of how cells efficiently direct receptors and their cargo to target destinations within the cytoplasm and at the plasma membrane. Specifically, light and 3D electron microscopy studies examining the trafficking behavior of the neonatal Fc receptor (FcRn), a transport receptor for immunoglobulin G (IgG), have given us new insights into the dynamic interplay between the structural components of the cytosolic trafficking machinery, its protein regulators, and the receptors it directs to various locations within the cell. These studies build upon previous biochemical characterizations of FcRn transport and are allowing us to begin formulation of a more complete model for the intracellular trafficking of receptor-ligand complexes.

  19. Melatonin receptor-mediated protection against myocardial ischaemia/reperfusion injury: role of its anti-adrenergic actions.

    Science.gov (United States)

    Genade, Sonia; Genis, Amanda; Ytrehus, Kirsti; Huisamen, Barbara; Lochner, Amanda

    2008-11-01

    Melatonin has potent cardioprotective properties. These actions have been attributed to its free radical scavenging and anti-oxidant actions, but may also be receptor mediated. Melatonin also exerts powerful anti-adrenergic actions based on its effects on contractility of isolated papillary muscles. The aims of this study were to determine whether melatonin also has anti-adrenergic effects on the isolated perfused rat heart, to determine the mechanism thereof and to establish whether these actions contribute to protection of the heart during ischaemia/reperfusion. The results showed that melatonin (50 microM) caused a significant reduction in both isoproterenol (10(-7) M) and forskolin (10(-6) M) induced cAMP production and that both these responses were melatonin receptor dependent, since the blocker, luzindole (5 x 10(-6) M) abolished this effect. Nitric oxide (NO), as well as guanylyl cyclase are involved, as L-NAME (50 microM), an NO synthase inhibitor and ODQ (20 microM), a guanylyl cyclase inhibitor, significantly counteracted the effects of melatonin. Protein kinase C (PKC), as indicated by the use of the inhibitor bisindolylmaleimide (50 microM), also play a role in melatonin's anti-adrenergic actions. These actions of melatonin are involved in its cardioprotection: simultaneous administration of L-NAME or ODQ with melatonin, before and after 35 min regional ischaemia, completely abolished its cardioprotection. PKC, on the other hand, had no effect on the melatonin-induced reduction in infarct size. Cardioprotection by melatonin was associated with a significant activation of PKB/Akt and attenuated activation of the pro-apoptotic kinase, p38MAPK during early reperfusion. In summary, the results show that melatonin-induced cardioprotection may be receptor dependent, and that its anti-adrenergic actions, mediated by NOS and guanylyl cyclase activation, are important contributors.

  20. Modulation of the input-output function by GABAA receptor-mediated currents in rat oculomotor nucleus motoneurons.

    Science.gov (United States)

    Torres-Torrelo, Julio; Torres, Blas; Carrascal, Livia

    2014-11-15

    The neuronal input-output function depends on recruitment threshold and gain of the firing frequency-current (f-I) relationship. These two parameters are positively correlated in ocular motoneurons (MNs) recorded in alert preparation and inhibitory inputs could contribute to this correlation. Phasic inhibition mediated by γ-amino butyric acid (GABA) occurs when a high concentration of GABA at the synaptic cleft activates postsynaptic GABAA receptors, allowing neuronal information transfer. In some neuronal populations, low concentrations of GABA activate non-synaptic GABAA receptors and generate a tonic inhibition, which modulates cell excitability. This study determined how ambient GABA concentrations modulate the input-output relationship of rat oculomotor nucleus MNs. Superfusion of brain slices with GABA (100 μm) produced a GABAA receptor-mediated current that reduced the input resistance, increased the recruitment threshold and shifted the f-I relationship rightward without any change in gain. These modifications did not depend on MN size. In absence of exogenous GABA, gabazine (20 μm; antagonist of GABAA receptors) abolished spontaneous inhibitory postsynaptic currents and revealed a tonic current in MNs. Gabazine increased input resistance and decreased recruitment threshold mainly in larger MNs. The f-I relationship shifted to the left, without any change in gain. Gabazine effects were chiefly due to MN tonic inhibition because tonic current amplitude was five-fold greater than phasic. This study demonstrates a tonic inhibition in ocular MNs that modulates cell excitability depending on cell size. We suggest that GABAA tonic inhibition acting concurrently with glutamate receptors activation could reproduce the positive covariation between threshold and gain reported in alert preparation.

  1. Long-term exposure to IL-1beta enhances Toll-IL-1 receptor-mediated inflammatory signaling in murine airway hyperresponsiveness

    DEFF Research Database (Denmark)

    Zhang, Yaping; Xu, Cang-Bao; Cardell, Lars-Olaf

    2009-01-01

    Toll-interleukin-1 (Toll-IL-1) receptor signaling may play a key role in the development of airway hyperreactivity (AHR) and chronic airway inflammatory diseases such as asthma. Previously, we have demonstrated that pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin......RNA expression following IL-1beta treatment. Immunohistochemistry confirmed that protein expression for CD14, RP105, MCP-1 and phosphorylated IkappaB-alpha were increased in both the airway epithelial and smooth muscle cells. In order to link the activation of Toll-IL-1 receptor-mediated inflammatory signal...... airway to IL-1beta induces up- and down-regulation of mRNA expression for Toll-IL-1 receptor signal molecules, with a significant increase in the expression of 16 genes that contribute to the development of airway inflammation and AHR. Understanding cytokine-induced activation of the Toll-IL-1 receptor...

  2. Receptor-mediated endocytosis and endosomal acidification is impaired in proximal tubule epithelial cells of Dent disease patients

    NARCIS (Netherlands)

    Gorvin, C.M.; Wilmer, M.J.G.; Piret, S.E.; Harding, B.; Heuvel, L.P.W.J. van den; Wrong, O.; Jat, P.S.; Lippiat, J.D.; Levtchenko, E.N.; Thakker, R.V.

    2013-01-01

    Receptor-mediated endocytosis, involving megalin and cubilin, mediates renal proximal-tubular reabsorption and is decreased in Dent disease because of mutations of the chloride/proton antiporter, chloride channel-5 (CLC-5), resulting in low-molecular-weight proteinuria, hypercalciuria, nephrolithias

  3. NK3 receptors mediate an increase in firing rate of midbrain dopamine neurons of the rat and the guinea pig.

    Science.gov (United States)

    Werkman, Taco R; McCreary, Andrew C; Kruse, Chris G; Wadman, Wytse J

    2011-08-01

    This in vitro study investigates and compares the effects of NK3 receptor ligands on the firing rate of rat and guinea pig midbrain dopamine neurons. The findings are discussed in the light of choosing suitable animal models for investigating pharmacological properties of NK3 receptor antagonists, which have been proposed to possess therapeutic activity in neuropsychiatric diseases like e.g. schizophrenia. In vitro midbrain slice preparations of both species were used to record (extracellularly) the firing rates of dopamine neurons located in the substantia nigra (SN) and ventral tegmental area (VTA). Furthermore, the effect of the D2 receptor agonist quinpirole on guinea pig SN and VTA dopamine neurons was investigated. The efficacy of quinpirole in inhibiting guinea pig dopamine neuron firing activity was much less as compared to that of rat dopamine neurons, suggesting a lower dopamine D2 autoreceptor density on the guinea pig neurons. The NK3 receptor agonist senktide induced in subpopulations of rat SN (55%) and VTA (79%) and guinea pig SN (50%) and VTA (21%) dopamine neurons an increase in firing rate. In responsive neurons this effect was concentration-dependent with EC₅₀ values of 3-5 nM (for both species). The selective NK3 receptor antagonist osanetant (100 nM) was able to partly block the senktide-induced increase in firing rates of dopamine neurons and shifted the concentration-response relation curves for senktide to the right (pA₂ values were ~7.5). The fractional block of the senktide responses by osanetant appeared to be larger in guinea pig dopamine neurons, indicating that osanetant is a more potent blocker of NK3 receptor-mediated responses with noncompetitive properties in the guinea pig.

  4. Differential modulation of expression of nuclear receptor mediated genes by tris(2-butoxyethyl) phosphate (TBOEP) on early life stages of zebrafish (Danio rerio)

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Zhiyuan, E-mail: zhiyuan_nju@163.com [State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Jiangsu 210023 (China); Yu, Yijun, E-mail: yjun.yu@gmail.com [State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Jiangsu 210023 (China); Tang, Song [School of Environment and Sustainability, University of Saskatchewan, Saskatoon, SK S7N 5B3 (Canada); Liu, Hongling, E-mail: hlliu@nju.edu.cn [State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Jiangsu 210023 (China); Su, Guanyong; Xie, Yuwei [State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Jiangsu 210023 (China); Giesy, John P. [State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Jiangsu 210023 (China); Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3 (Canada); Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK S7N 5B3 (Canada); Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong Special Administrative Region (Hong Kong); Hecker, Markus [School of Environment and Sustainability, University of Saskatchewan, Saskatoon, SK S7N 5B3 (Canada); Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3 (Canada); Yu, Hongxia [State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Jiangsu 210023 (China)

    2015-12-15

    Highlights: • Effects of TBOEP on expression of genes of several nuclear hormone receptors and their relationship with adverse effect pathways in zebrafish. • TBOEP was neither an agonist nor antagonist of AR or AhR as determined by use of in vitro mammalian cell-based receptor transactivation assays. • Modulation of ER- and MR-dependent pathways allowed for development of feasible receptor-mediated, critical mechanisms of toxic action. - Abstract: As one substitute for phased-out brominated flame retardants (BFRs), tris(2-butoxyethyl) phosphate (TBOEP) is frequently detected in aquatic organisms. However, knowledge about endocrine disrupting mechanisms associated with nuclear receptors caused by TBOEP remained restricted to results from in vitro studies with mammalian cells. In the study, results of which are presented here, embryos/larvae of zebrafish (Danio rerio) were exposed to 0.02, 0.1 or 0.5 μM TBOEP to investigate expression of genes under control of several nuclear hormone receptors (estrogen receptors (ERs), androgen receptor (AR), thyroid hormone receptor alpha (TRα), mineralocorticoid receptor (MR), glucocorticoid receptor (GR), aryl hydrocarbon (AhR), peroxisome proliferator-activated receptor alpha (PPARα), and pregnane × receptor (P × R)) pathways at 120 hpf. Exposure to 0.5 μM TBOEP significantly (p < 0.05, one-way analysis of variance) up-regulated expression of estrogen receptors (ERs, er1, er2a, and er2b) genes and ER-associated genes (vtg4, vtg5, pgr, ncor, and ncoa3), indicating TBOEP modulates the ER pathway. In contrast, expression of most genes (mr, 11βhsd, ube2i,and adrb2b) associated with the mineralocorticoid receptor (MR) pathway were significantly down-regulated. Furthermore, in vitro mammalian cell-based (MDA-kb2 and H4IIE-luc) receptor transactivation assays, were also conducted to investigate possible agonistic or antagonistic effects on AR- and AhR-mediated pathways. In mammalian cells, none of these pathways were

  5. [Properties of cholinergic receptor-mediated ion channels on type I vestibular hair cells of guinea pigs].

    Science.gov (United States)

    Zhu, Yun; Kong, Wei-Jia; Xia, Jiao; Zhang, Yu; Cheng, Hua-Mao; Guo, Chang-Kai

    2008-06-25

    To confirm the existence of cholinergic receptors on type I vestibular hair cells (VHCs I) of guinea pigs and to study the properties of the cholinergic receptor-mediated ion channels on VHCs I, electrophysiological responses of isolated VHCs I to external ACh were examined by means of whole-cell patch-clamp recordings. The results showed that 7.5% (21/279) VHCs I were found to be sensitive to ACh (10-1000 μmol/L). ACh generated an outward current in a steady, slow, dose-dependent [EC(50) was (63.78±2.31) μmol/L] and voltage-independent manner. In standard extracellular solution, ACh at the concentration of 100 μmol/L triggered a calcium-dependent current of (170±15) pA at holding potential of -50 mV, and the current amplitude could be depressed by extracellularly added calcium-dependent potassium channel antagonist TEA. The time interval for the next complete activation of ACh-sensitive current was no less than 1 min. The ion channels did not shut off even when they were exposed to ACh for an extended period of time (8 min). The results suggest that dose-dependent, calcium-dependent and voltage-independent cholinergic receptors were located on a few of the VHCs I investibular epithelium of guinea pigs. The cholinergic receptors did not show desensitization to ACh. This work reveals the existence of efferent neurotransmitter receptors on VHCs I and helps in understanding the function of vestibular efferent nervous system, and may provide some useful information on guiding the clinical rehabilitative treatment of vertigo.

  6. Exploring the contextual sensitivity of factors that determine cell-to-cell variability in receptor-mediated apoptosis.

    Directory of Open Access Journals (Sweden)

    Suzanne Gaudet

    Full Text Available Stochastic fluctuations in gene expression give rise to cell-to-cell variability in protein levels which can potentially cause variability in cellular phenotype. For TRAIL (TNF-related apoptosis-inducing ligand variability manifests itself as dramatic differences in the time between ligand exposure and the sudden activation of the effector caspases that kill cells. However, the contribution of individual proteins to phenotypic variability has not been explored in detail. In this paper we use feature-based sensitivity analysis as a means to estimate the impact of variation in key apoptosis regulators on variability in the dynamics of cell death. We use Monte Carlo sampling from measured protein concentration distributions in combination with a previously validated ordinary differential equation model of apoptosis to simulate the dynamics of receptor-mediated apoptosis. We find that variation in the concentrations of some proteins matters much more than variation in others and that precisely which proteins matter depends both on the concentrations of other proteins and on whether correlations in protein levels are taken into account. A prediction from simulation that we confirm experimentally is that variability in fate is sensitive to even small increases in the levels of Bcl-2. We also show that sensitivity to Bcl-2 levels is itself sensitive to the levels of interacting proteins. The contextual dependency is implicit in the mathematical formulation of sensitivity, but our data show that it is also important for biologically relevant parameter values. Our work provides a conceptual and practical means to study and understand the impact of cell-to-cell variability in protein expression levels on cell fate using deterministic models and sampling from parameter distributions.

  7. Testin, a novel binding partner of the calcium-sensing receptor, enhances receptor-mediated Rho-kinase signalling

    Energy Technology Data Exchange (ETDEWEB)

    Magno, Aaron L. [Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, Nedlands, Western Australia 6009 (Australia); Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, Western Australia 6009 (Australia); Ingley, Evan [Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, Nedlands, Western Australia 6009 (Australia); Brown, Suzanne J. [Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, Western Australia 6009 (Australia); Conigrave, Arthur D. [School of Molecular Bioscience, University of Sydney, New South Wales 2000 (Australia); Ratajczak, Thomas [Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, Nedlands, Western Australia 6009 (Australia); Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, Western Australia 6009 (Australia); Ward, Bryan K., E-mail: bryanw@cyllene.uwa.edu.au [Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, Nedlands, Western Australia 6009 (Australia); Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, Western Australia 6009 (Australia)

    2011-09-09

    Highlights: {yields} A yeast two-hybrid screen revealed testin bound to the calcium-sensing receptor. {yields} The second zinc finger of LIM domain 1 of testin is critical for interaction. {yields} Testin bound to a region of the receptor tail important for cell signalling. {yields} Testin and receptor interaction was confirmed in mammalian (HEK293) cells. {yields} Overexpression of testin enhanced receptor-mediated Rho signalling in HEK293 cells. -- Abstract: The calcium-sensing receptor (CaR) plays an integral role in calcium homeostasis and the regulation of other cellular functions including cell proliferation and cytoskeletal organisation. The multifunctional nature of the CaR is manifested through ligand-dependent stimulation of different signalling pathways that are also regulated by partner binding proteins. Following a yeast two-hybrid library screen using the intracellular tail of the CaR as bait, we identified several novel binding partners including the focal adhesion protein, testin. Testin has not previously been shown to interact with cell surface receptors. The sites of interaction between the CaR and testin were mapped to the membrane proximal region of the receptor tail and the second zinc-finger of LIM domain 1 of testin, the integrity of which was found to be critical for the CaR-testin interaction. The CaR-testin association was confirmed in HEK293 cells by coimmunoprecipitation and confocal microscopy studies. Ectopic expression of testin in HEK293 cells stably expressing the CaR enhanced CaR-stimulated Rho activity but had no effect on CaR-stimulated ERK signalling. These results suggest an interplay between the CaR and testin in the regulation of CaR-mediated Rho signalling with possible effects on the cytoskeleton.

  8. Interferon-α/β receptor-mediated selective induction of a gene cluster by CpG oligodeoxynucleotide 2006

    Directory of Open Access Journals (Sweden)

    Wakiguchi Hiroshi

    2003-07-01

    Full Text Available Abstract Background Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN are known to exert a strong adjuvant effect on Th1 immune responses. Although several genes have been reported, no comprehensive study of the gene expression profiles in human cells after stimulation with CpG ODN has been reported. Results This study was designed to identify a CpG-inducible gene cluster that potentially predicts for the molecular mechanisms of clinical efficacy of CpG ODN, by determining mRNA expression in human PBMC after stimulation with CpG ODN. PBMCs were obtained from the peripheral blood of healthy volunteers and cultured in the presence or absence of CpG ODN 2006 for up to 24 hours. The mRNA expression profile was evaluated using a high-density oligonucleotide probe array, GeneChip®. Using hierarchical clustering-analysis, out of a total of 10,000 genes we identified a cluster containing 77 genes as having been up-regulated by CpG ODN. This cluster was further divided into two sub-clusters by means of time-kinetics. (1 Inflammatory cytokines such as IL-6 and GM-CSF were up-regulated predominantly 3 to 6 hours after stimulation with CpG ODN, presumably through activation of a transcription factor, NF-κB. (2 Interferon (IFN-inducible anti-viral proteins, including IFIT1, OAS1 and Mx1, and Th1 chemoattractant IP-10, were up-regulated predominantly 6 to 24 hours after stimulation. Blocking with mAb against IFN-α/β receptor strongly inhibited the induction of these IFN-inducible genes by CpG ODN. Conclusion This study provides new information regarding the possible immunomodulatory effects of CpG ODN in vivo via an IFN-α/β receptor-mediated paracrine pathway.

  9. Ontogeny of catecholamine and adenosine receptor-mediated cAMP signaling of embryonic red blood cells: role of cGMP-inhibited phosphodiesterase 3 and hemoglobin.

    Science.gov (United States)

    Baumann, R; Blass, C; Götz, R; Dragon, S

    1999-12-15

    We have previously shown that the cAMP signaling pathway controls major aspects of embryonic red blood cell (RBC) function in avian embryos (Glombitza et al, Am J Physiol 271:R973, 1996; and Dragon et al, Am J Physiol 271:R982, 1996) that are important for adaptation of the RBC gas transport properties to the progressive hypercapnia and hypoxia of later stages of avian embryonic development. Data about the ontogeny of receptor-mediated cAMP signaling are lacking. We have analyzed the response of primitive and definitive chick embryo RBC harvested from day 3 to 18 of development towards forskolin, beta-adrenergic, and A2 receptor agonists. The results show a strong response of immature definitive and primitive RBC to adenosine A2 and beta-adrenergic receptor agonists, which is drastically reduced in the last stage of development, coincident with the appearance of mature, transcriptionally inactive RBC. Modulation of cGMP-inhibited phosphodiesterase 3 (PDE3) has a controlling influence on cAMP accumulation in definitive RBC. Under physiological conditions, PDE3 is inhibited due to activation of soluble guanylyl cyclase (sGC). Inhibition of sGC with the specific inhibitor ODQ decreases receptor-mediated stimulation of cAMP production; this effect is reversed by the PDE3 inhibitor milrinone. sGC is acitivated by nitric oxide (NO), but we found no evidence for production of NO by erythrocyte NO-synthase. However, embryonic hemoglobin releases NO in an oxygen-linked manner that may activate guanylyl cyclase.

  10. Nicotine impairs cyclooxygenase-2-dependent kinin-receptor-mediated murine airway relaxations

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yuan, E-mail: yuan.xu@ki.se; Cardell, Lars-Olaf

    2014-02-15

    Introduction: Cigarette smoke induces local inflammation and airway hyperreactivity. In asthmatics, it worsens the symptoms and increases the risk for exacerbation. The present study investigates the effects of nicotine on airway relaxations in isolated murine tracheal segments. Methods: Segments were cultured for 24 h in the presence of vehicle, nicotine (10 μM) and/or dexamethasone (1 μM). Airway relaxations were assessed in myographs after pre-contraction with carbachol (1 μM). Kinin receptors, cyclooxygenase (COX) and inflammatory mediator expressions were assessed by real-time PCR and confocal-microscopy-based immunohistochemistry. Results: The organ culture procedure markedly increased bradykinin- (selective B{sub 2} receptor agonist) and des-Arg{sup 9}-bradykinin- (selective B{sub 1} receptor agonist) induced relaxations, and slightly increased relaxation induced by isoprenaline, but not that induced by PGE{sub 2}. The kinin receptor mediated relaxations were epithelium-, COX-2- and EP2-receptor-dependent and accompanied by drastically enhanced mRNA levels of kinin receptors, as well as inflammatory mediators MCP-1 and iNOS. Increase in COX-2 and mPGES-1 was verified both at mRNA and protein levels. Nicotine selectively suppressed the organ-culture-enhanced relaxations induced by des-Arg{sup 9}-bradykinin and bradykinin, at the same time reducing mPGES-1 mRNA and protein expressions. α7-nicotinic acetylcholine receptor inhibitors α-bungarotoxin and MG624 both blocked the nicotine effects on kinin B{sub 2} receptors, but not those on B{sub 1}. Dexamethasone completely abolished kinin-induced relaxations. Conclusion: It is tempting to conclude that a local inflammatory process per se could have a bronchoprotective component by increasing COX-2 mediated airway relaxations and that nicotine could impede this safety mechanism. Dexamethasone further reduced airway inflammation together with relaxations. This might contribute to the steroid resistance seen in

  11. H2 receptor-mediated facilitation and H3 receptor-mediated inhibition of noradrenaline release in the guinea-pig brain.

    Science.gov (United States)

    Timm, J; Marr, I; Werthwein, S; Elz, S; Schunack, W; Schlicker, E

    1998-03-01

    , hippocampal or hypothalamic slices were used instead of cortical slices. The Ca2+-induced tritium overflow in guinea-pig cortex slices was inhibited by histamine (in the presence of ranitidine); this effect was abolished by clobenpropit. In slices superfused in the presence of clobenpropit, impromidine failed to facilitate the Ca2+-evoked tritium overflow. The electrically evoked tritium overflow in mouse brain cortex slices was inhibited by histamine by about 60% (both in the absence or presence of ranitidine). The inhibitory effect of histamine was abolished (but not reversed) by clobenpropit. In conclusion, noradrenaline release in the guinea-pig brain cortex is inhibited via presynaptic H3 receptors and facilitated via H2 receptors not located presynaptically. In the mouse brain cortex, only inhibitory H3 receptors occur. The extent of the H3 receptor-mediated effect is more marked in the mouse than in the guinea-pig brain cortex.

  12. Multivalent ligand-receptor-mediated interaction of small filled vesicles with a cellular membrane

    Science.gov (United States)

    Zhdanov, Vladimir P.

    2017-07-01

    The ligand-receptor-mediated contacts of small sub-100-nm-sized lipid vesicles (or nanoparticles) with the cellular membrane are of interest in the contexts of cell-to-cell communication, endocytosis of membrane-coated virions, and drug (RNA) delivery. In all these cases, the interior of vesicles is filled by biologically relevant content. Despite the diversity of such systems, the corresponding ligand-receptor interaction possesses universal features. One of them is that the vesicle-membrane contacts can be accompanied by the redistribution of ligands and receptors between the contact and contact-free regions. In particular, the concentrations of ligands and receptors may become appreciably higher in the contact regions and their composition may there be different compared to that in the suspended state in the solution. A statistical model presented herein describes the corresponding distribution of various ligands and receptors and allows one to calculate the related change of the free energy with variation of the vesicle-engulfment extent. The results obtained are used to clarify the necessary conditions for the vesicle-assisted pathway of drug delivery.

  13. Kainate receptors mediate signaling in both transient and sustained OFF bipolar cell pathways in mouse retina.

    Science.gov (United States)

    Borghuis, Bart G; Looger, Loren L; Tomita, Susumu; Demb, Jonathan B

    2014-04-30

    A fundamental question in sensory neuroscience is how parallel processing is implemented at the level of molecular and circuit mechanisms. In the retina, it has been proposed that distinct OFF cone bipolar cell types generate fast/transient and slow/sustained pathways by the differential expression of AMPA- and kainate-type glutamate receptors, respectively. However, the functional significance of these receptors in the intact circuit during light stimulation remains unclear. Here, we measured glutamate release from mouse bipolar cells by two-photon imaging of a glutamate sensor (iGluSnFR) expressed on postsynaptic amacrine and ganglion cell dendrites. In both transient and sustained OFF layers, cone-driven glutamate release from bipolar cells was blocked by antagonists to kainate receptors but not AMPA receptors. Electrophysiological recordings from bipolar and ganglion cells confirmed the essential role of kainate receptors for signaling in both transient and sustained OFF pathways. Kainate receptors mediated responses to contrast modulation up to 20 Hz. Light-evoked responses in all mouse OFF bipolar pathways depend on kainate, not AMPA, receptors.

  14. Receptor-mediated endocytosis of lysozyme in renal proximal tubules of the frog Rana temporaria

    Directory of Open Access Journals (Sweden)

    E.V. Seliverstova

    2015-04-01

    Full Text Available The mechanism of protein reabsorption in the kidney of lower vertebrates remains insufficiently investigated in spite of raising interest to the amphibian and fish kidneys as a useful model for physiological and pathophysiological examinations. In the present study, we examined the renal tubular uptake and the internalization rote of lysozyme after its intravenous injection in the wintering frog Rana temporaria using immunohisto- and immunocytochemistry and specific markers for some endocytic compartments. The distinct expression of megalin and cubilin in the proximal tubule cells of lysozyme-injected frogs was revealed whereas kidney tissue of control animals showed no positive immunoreactivity. Lysozyme was detected in the apical endocytic compartment of the tubular cells and colocalized with clathrin 10 min after injection. After 20 min, lysozyme was located in the subapical compartment negative to clathrin (endosomes, and intracellular trafficking of lysozyme was coincided with the distribution of megalin and cubilin. However, internalized protein was retained in the endosomes and did not reach lysosomes within 30 min after treatment that may indicate the inhibition of intracellular trafficking in hibernating frogs. For the first time, we provided the evidence that lysozyme is filtered through the glomeruli and absorbed by receptor-mediated clathrin-dependent endocytosis in the frog proximal tubule cells. Thus, the protein uptake in the amphibian mesonephros is mediated by megalin and cubilin that confirms a critical role of endocytic receptors in the renal reabsorption of proteins in amphibians as in mammals.

  15. A new Kupffer cell receptor mediating plasma clearance of carcinoembryonic antigen by the rat.

    Science.gov (United States)

    Toth, C A; Thomas, P; Broitman, S A; Zamcheck, N

    1982-05-15

    Native human carcinoembryonic antigen is rapidly removed from the circulation by the rat liver Kupffer cell after intravenous injection. The molecule is subsequently transferred to the hepatocyte in an immunologically identifiable form. Carcinoembryonic antigen has a circulatory half-life of 3.7 (+/- 0.8) min, and cellular entry is by receptor-mediated endocytosis. Non-specific fluid pinocytosis and phagocytosis can be excluded as possible mechanisms by the kinetics of clearance and failure of colloidal carbon to inhibit uptake. Substances with known affinity for the hepatic receptors for mannose, N-acetylglucosamine, fucose and galactose all fail to inhibit carcinoembryonic antigen clearance. After two cycles of the Smith degradation, carcinoembryonic antigen is still able to inhibit clearance of the native molecule. Receptor specificity is apparently not dependent on those non-reducing terminal sugars of the native molecule. Performic acid-oxidized carcinoembryonic antigen also inhibits clearance of carcinoembryonic antigen in vivo. Receptor binding is not dependent on tertiary protein conformation. Non-specific cross-reacting antigen, a glycoprotein structurally similar to carcinoembryonic antigen, is cleared by the same mechanism.

  16. Receptor-Mediated and Fluid-Phase Transcytosis of Horseradish Peroxidase across Rat Hepatocytes

    Directory of Open Access Journals (Sweden)

    Isabella Ellinger

    2010-01-01

    Full Text Available Horseradish peroxidase (HRP is often used as a fluid-phase marker to characterize endocytic and transcytotic processes. Likewise, it has been applied to investigate the mechanisms of biliary secretion of fluid in rat liver hepatocytes. However, HRP contains mannose residues and thus binds to mannose receptors (MRs on liver cells, including hepatocytes. To study the role of MR-mediated endocytosis of HRP transport in hepatocytes, we determined the influence of the oligosaccharid mannan on HRP biliary secretion in the isolated perfused rat liver. A 1-minute pulse of HRP was applied followed by marker-free perfusion. HRP appeared in bile with biphasic kinetics: a first peak at 7 minutes and a second peak at 15 minutes after labeling. Perfusion with 0.8 mg/mL HRP in the presence of a twofold excess of mannan reduced the first peak by 41% without effect on the second one. Together with recently published data on MR expression in rat hepatocytes this demonstrates two different mechanisms for HRP transcytosis: a rapid, receptor-mediated transport and a slower fluid-phase transport.

  17. ARRHYTHMOGENIC CALMODULIN MUTATIONS AFFECT THE ACTIVATION AND TERMINATION OF CARDIAC RYANODINE RECEPTOR MEDIATED CA2+ RELEASE

    DEFF Research Database (Denmark)

    Søndergaard, Mads Toft; Chazin, Walter J.; Chen, Wayne S.R.;

    We recently identified the first two human missense mutations in a calmodulin (CaM) gene (CALM1) and linked these to catecholaminergic polymorphic ventricular tachycardia (CPVT) and sudden cardiac death in young individuals1. More CaM mutations have since been identified in CALM1 and also......M in the presence of RyR2 CaMBD. The D95V, N97S and D129G mutations lowered the affinity of Ca2+ binding of the C-lobe of CaM, to apparent KDs of ~ 140, 150, and 4000 nM, respectively, consistent with the critical role of these residues in Ca2+ binding to the C-lobe. Thus, we suggest that these mutations may shift...... to an apo-CaM binding state during diastole, leading to dysregulation of RyR2 mediated Ca2+ release. Despite the pronounced impact on RyR2 mediated Ca2+ release, the N-lobe N53I mutation only imposed a small lowering of the N-lobe Ca2+ affinity (KD ~1200 nM). Thus, the RyR2 mediated Ca2+ release is either...

  18. Modeling Nuclear Receptor-Mediated Activity and Hepatotoxicity with Boolean Networks

    Science.gov (United States)

    Predicting the human health risk of chronic exposure to environmental contaminants remains an open problem. Chronic exposure to a wide array of chemicals – e.g., conazoles, perfluourinated chemicals and phthalates – has been associated with a range of hepatic lesions in rodents t...

  19. Modulation of Toll-like receptor-mediated activation of Microglia

    NARCIS (Netherlands)

    Putten, C. M.-T. van der

    2015-01-01

    Microglia are the resident macrophages of the central nervous system (CNS). Like other tissue macrophages, microglia have many different functions under physiological as well as pathological conditions. Microglia can contribute to the initiation, progression and resolution of disease processes and m

  20. Toll-like receptor mediated activation is possibly involved in immunoregulating properties of cow's milk hydrolysates

    NARCIS (Netherlands)

    Kiewiet, M. B. Gea; Dekkers, Renske; Gros, Marjan; van Neerven, R. J. Joost; Groeneveld, Andre; de Vos, Paul; Faas, Marijke M.

    2017-01-01

    Immunomodulating proteins and peptides are formed during the hydrolysis of cow's milk proteins. These proteins are potential ingredients in functional foods used for the management of a range of immune related problems, both in infants and adults. However, the mechanism behind these effects is

  1. GABAA Receptor-Mediated Activity in a Model of Cortical Dysplasia

    Science.gov (United States)

    2012-06-29

    epilepsy, autism and schizophrenia, and results from failure of immature neurons to appropriately migrate to their cortical targets. We developed a...schizophrenia, and autism , and results from failure of immature neurons to appropriately migrate and reach their cortical targets (Taylor et al., 1971; Choi and...appropriate cortical target is regulated by Brn1 and Brn2, POU-domain transcription factors that regulate the reelin /DAB1 pathway, and cyclin

  2. Hypersensitivity to thromboxane receptor mediated cerebral vasomotion and CBF oscillations during acute NO-deficiency in rats.

    Directory of Open Access Journals (Sweden)

    Béla Horváth

    Full Text Available BACKGROUND: Low frequency (4-12 cpm spontaneous fluctuations of the cerebrovascular tone (vasomotion and oscillations of the cerebral blood flow (CBF have been reported in diseases associated with endothelial dysfunction. Since endothelium-derived nitric oxide (NO suppresses constitutively the release and vascular effects of thromboxane A(2 (TXA(2, NO-deficiency is often associated with activation of thromboxane receptors (TP. In the present study we hypothesized that in the absence of NO, overactivation of the TP-receptor mediated cerebrovascular signaling pathway contributes to the development of vasomotion and CBF oscillations. METHODOLOGY/PRINCIPAL FINDINGS: Effects of pharmacological modulation of TP-receptor activation and its downstream signaling pathway have been investigated on CBF oscillations (measured by laser-Doppler flowmetry in anesthetized rats and vasomotion (measured by isometric tension recording in isolated rat middle cerebral arteries, MCAs both under physiological conditions and after acute inhibition of NO synthesis. Administration of the TP-receptor agonist U-46619 (1 µg/kg i.v. to control animals failed to induce any changes of the systemic or cerebral circulatory parameters. Inhibition of the NO synthesis by nitro-L-arginine methyl ester (L-NAME, 100 mg/kg i.v. resulted in increased mean arterial blood pressure and a decreased CBF accompanied by appearance of CBF-oscillations with a dominant frequency of 148±2 mHz. U-46619 significantly augmented the CBF-oscillations induced by L-NAME while inhibition of endogenous TXA(2 synthesis by ozagrel (10 mg/kg i.v. attenuated it. In isolated MCAs U-46619 in a concentration of 100 nM, which induced weak and stable contraction under physiological conditions, evoked sustained vasomotion in the absence of NO, which effect could be completely reversed by inhibition of Rho-kinase by 10 µM Y-27632. CONCLUSION/SIGNIFICANCE: These results suggest that hypersensitivity of the TP

  3. α1A-adrenergic receptor mediated pressor response to phenylephrine in anesthetized rat

    Institute of Scientific and Technical Information of China (English)

    XU Qi; ZHU Weizhong; L(U) Zhizhen; ZHANG Youyi; HAN Qide

    2004-01-01

    To determine which subtype of α1A-adrenergic receptors plays a role in the regulation of blood pressure, with α1A-adrenergic receptor-mediated vasoconstriction in perfused hindlimb as a control, we compared the inhibitory effects of various α1A-adrenergic receptor selective antagonists on the vasopressure responses to phenylephrine between the mean arterial pressure and hindlimb perfusion pressure in anesthetized rats. In Normotensive Wistar rats, the results showed that the inhibitory effects (dose ratios of ED50, Dr) of α1A-adrenoceptor selective antagonist (prazosin, Dr 13.5 ± 3.6 vs.15.1 ± 4.3, n = 11), α1A-adrenoceptor selective antagonist (5- methyl-urapidil, Dr 2.4 ± 0.9 vs. 3.7 ± 2.3, n = 12; RS-17053, Dr 3.2 ± 1.6 vs. 4.4 ± 3.3, n =12) and α1D- adrenoceptor selective antagonist (BMY7378, Dr 1.9 ± 0.9 vs. 2.2 ± 0.8, n = 8) on phenylephrine- induced increases of perfusion pressure in the autoperfused femoral beds were the same as that in the mean arterial blood pressure in normotensive Wistar rats. The inhibitory effects of antagonists (RS-17053, Dr 3.4 ± 0.6 vs. 4.3 ± 0.9, n = 5; BMY7378, Dr 1.7±0.5 vs. 1.7 ± 0.5, n = 8) in spontaneous hypertensive rats were similar with the Wistar rats. These results suggest that the mean arterial pressure induced by phenylephrine was mainly mediated by α1A-adrenergic receptor in both the anesthetized Wistar rats and spontaneous hypertensive rats.

  4. EP2 receptor mediates PGE2-induced cystogenesis of human renal epithelial cells.

    Science.gov (United States)

    Elberg, Gerard; Elberg, Dorit; Lewis, Teresa V; Guruswamy, Suresh; Chen, Lijuan; Logan, Charlotte J; Chan, Michael D; Turman, Martin A

    2007-11-01

    Autosomal-dominant polycystic kidney disease (ADPKD) is characterized by formation of cysts from tubular epithelial cells. Previous studies indicate that secretion of prostaglandin E2 (PGE2) into cyst fluid and production of cAMP underlie cyst expansion. However, the mechanism by which PGE2 directly stimulates cAMP formation and modulates cystogenesis is still unclear, because the particular E-prostanoid (EP) receptor mediating the PGE2 effect has not been characterized. Our goal is to define the PGE2 receptor subtype involved in ADPKD. We used a three-dimensional cell-culture system of human epithelial cells from normal and ADPKD kidneys in primary cultures to demonstrate that PGE2 induces cyst formation. Biochemical evidence gathered by using real-time RT-PCR mRNA analysis and immunodetection indicate the presence of EP2 receptor in cystic epithelial cells in ADPKD kidney. Pharmacological evidence obtained by using PGE2-selective analogs further demonstrates that EP2 mediates cAMP formation and cystogenesis. Functional evidence for a role of EP2 receptor in mediating cAMP signaling was also provided by inhibiting EP2 receptor expression with transfection of small interfering RNA in cystic epithelial cells. Our results indicate that PGE2 produced in cyst fluid binds to adjacent EP2 receptors located on the apical side of cysts and stimulates EP2 receptor expression. PGE2 binding to EP2 receptor leads to cAMP signaling and cystogenesis by a mechanism that involves protection of cystic epithelial cells from apoptosis. The role of EP2 receptor in mediating the PGE2 effect on stimulating cyst formation may have direct pharmacological implications for the treatment of polycystic kidney disease.

  5. Scavenger receptors mediate the role of SUMO and Ftz-f1 in Drosophila steroidogenesis.

    Directory of Open Access Journals (Sweden)

    Ana Talamillo

    2013-04-01

    Full Text Available SUMOylation participates in ecdysteroid biosynthesis at the onset of metamorphosis in Drosophila melanogaster. Silencing the Drosophila SUMO homologue smt3 in the prothoracic gland leads to reduced lipid content, low ecdysone titers, and a block in the larval-pupal transition. Here we show that the SR-BI family of Scavenger Receptors mediates SUMO functions. Reduced levels of Snmp1 compromise lipid uptake in the prothoracic gland. In addition, overexpression of Snmp1 is able to recover lipid droplet levels in the smt3 knockdown prothoracic gland cells. Snmp1 expression depends on Ftz-f1 (an NR5A-type orphan nuclear receptor, the expression of which, in turn, depends on SUMO. Furthermore, we show by in vitro and in vivo experiments that Ftz-f1 is SUMOylated. RNAi-mediated knockdown of ftz-f1 phenocopies that of smt3 at the larval to pupal transition, thus Ftz-f1 is an interesting candidate to mediate some of the functions of SUMO at the onset of metamorphosis. Additionally, we demonstrate that the role of SUMOylation, Ftz-f1, and the Scavenger Receptors in lipid capture and mobilization is conserved in other steroidogenic tissues such as the follicle cells of the ovary. smt3 knockdown, as well as ftz-f1 or Scavenger knockdown, depleted the lipid content of the follicle cells, which could be rescued by Snmp1 overexpression. Therefore, our data provide new insights into the regulation of metamorphosis via lipid homeostasis, showing that Drosophila Smt3, Ftz-f1, and SR-BIs are part of a general mechanism for uptake of lipids such as cholesterol, required during development in steroidogenic tissues.

  6. 5-HT(1A) and 5-HT(7) receptors differently modulate AMPA receptor-mediated hippocampal synaptic transmission.

    Science.gov (United States)

    Costa, L; Trovato, C; Musumeci, S A; Catania, M V; Ciranna, L

    2012-04-01

    We have studied the effects of 5-HT(1A) and 5-HT(7) serotonin receptor activation in hippocampal CA3-CA1 synaptic transmission using patch clamp on mouse brain slices. Application of either 5-HT or 8-OH DPAT, a mixed 5-HT(1A)/5-HT(7) receptor agonist, inhibited AMPA receptor-mediated excitatory post synaptic currents (EPSCs); this effect was mimicked by the 5-HT(1A) receptor agonist 8-OH PIPAT and blocked by the 5-HT(1A) antagonist NAN-190. 8-OH DPAT increased paired-pulse facilitation and reduced the frequency of mEPSCs, indicating a presynaptic reduction of glutamate release probability. In another group of neurons, 8-OH DPAT enhanced EPSC amplitude but did not alter paired-pulse facilitation, suggesting a postsynaptic action; this effect persisted in the presence of NAN-190 and was blocked by the 5-HT(7) receptor antagonist SB-269970. To confirm that EPSC enhancement was mediated by 5-HT(7) receptors, we used the compound LP-44, which is considered a selective 5-HT(7) agonist. However, LP-44 reduced EPSC amplitude in most cells and instead increased EPSC amplitude in a subset of neurons, similarly to 8-OH DPAT. These effects were respectively antagonized by NAN-190 and by SB-269970, indicating that under our experimental condition LP-44 behaved as a mixed agonist. 8-OH DPAT also modulated the current evoked by exogenously applied AMPA, inducing either a reduction or an increase of amplitude in distinct neurons; these effects were respectively blocked by 5-HT(1A) and 5-HT(7) receptor antagonists, indicating that both receptors exert a postsynaptic action. Our results show that 5-HT(1A) receptors inhibit CA3-CA1 synaptic transmission acting both pre- and postsynaptically, whereas 5-HT(7) receptors enhance CA3-CA1 synaptic transmission acting exclusively at a postsynaptic site. We suggest that a selective pharmacological targeting of either subtype may be envisaged in pathological loss of hippocampal-dependent cognitive functions. In this respect, we underline the

  7. Synaptic NMDA receptor-mediated currents in anterior piriform cortex are reduced in the adult fragile X mouse.

    Science.gov (United States)

    Gocel, James; Larson, John

    2012-09-27

    Fragile X syndrome is a neurodevelopmental condition caused by the transcriptional silencing of the fragile X mental retardation 1 (FMR1) gene. The Fmr1 knockout (KO) mouse exhibits age-dependent deficits in long term potentiation (LTP) at association (ASSN) synapses in anterior piriform cortex (APC). To investigate the mechanisms for this, whole-cell voltage-clamp recordings of ASSN stimulation-evoked synaptic currents were made in APC of slices from adult Fmr1-KO and wild-type (WT) mice, using the competitive N-methyl-D-aspartate (NMDA) receptor antagonist, CPP, to distinguish currents mediated by NMDA and AMPA receptors. NMDA/AMPA current ratios were lower in Fmr1-KO mice than in WT mice, at ages ranging from 3-18months. Since amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs) mediated by AMPA receptors were no different in Fmr1-KO and WT mice at these ages, the results suggest that NMDA receptor-mediated currents are selectively reduced in Fmr1-KO mice. Analyses of voltage-dependence and decay kinetics of NMDA receptor-mediated currents did not reveal differences between Fmr1-KO and WT mice, suggesting that reduced NMDA currents in Fmr1-KO mice are due to fewer synaptic receptors rather than differences in receptor subunit composition. Reduced NMDA receptor signaling may help to explain the LTP deficit seen at APC ASSN synapses in Fmr1-KO mice at 6-18months of age, but does not explain normal LTP at these synapses in mice 3-6months old. Evoked currents and mEPSCs were also examined in senescent Fmr1-KO and WT mice at 24-28months of age. NMDA/AMPA ratios were similar in senescent WT and Fmr1-KO mice, due to a decrease in the ratio in the WT mice, without significant change in AMPA receptor-mediated mEPSCs.

  8. Specific Endocytosis Blockade of Trypanosoma cruzi Exposed to a Poly-LAcNAc Binding Lectin Suggests that Lectin-Sugar Interactions Participate to Receptor-Mediated Endocytosis

    Science.gov (United States)

    Brosson, Sébastien; Fontaine, Frédéric; Vermeersch, Marjorie; Perez-Morga, David; Pays, Etienne; Bousbata, Sabrina; Salmon, Didier

    2016-01-01

    Trypanosoma cruzi is a protozoan parasite transmitted by a triatomine insect, and causing human Chagas disease in South America. This parasite undergoes a complex life cycle alternating between non-proliferative and dividing forms. Owing to their high energy requirement, replicative epimastigotes of the insect midgut display high endocytic activity. This activity is mainly restricted to the cytostome, by which the cargo is taken up and sorted through the endosomal vesicular network to be delivered to reservosomes, the final lysosomal-like compartments. In African trypanosomes tomato lectin (TL) and ricin, respectively specific to poly-N-acetyllactosamine (poly-LacNAc) and β-D-galactose, allowed the identification of giant chains of poly-LacNAc in N-glycoproteins of the endocytic pathway. We show that in T. cruzi epimastigote forms also, glycoproteins of the endocytic pathway are characterized by the presence of N-linked glycans binding to both ricin and TL. Affinity chromatography using both TL and Griffonia simplicifolia lectin II (GSLII), specific to non-reducing terminal residue of N-acetylglucosamine (GlcNAc), led to an enrichment of glycoproteins of the trypanosomal endocytic pathway. Incubation of live parasites with TL, which selectively bound to the cytostome/cytopharynx, specifically inhibited endocytosis of transferrin (Tf) but not dextran, a marker of fluid endocytosis. Taken together, our data suggest that N-glycan modification of endocytic components plays a crucial role in receptor-mediated endocytosis of T. cruzi. PMID:27685262

  9. Luteolin Reduces BACE1 Expression through NF-κB and through Estrogen Receptor Mediated Pathways in HEK293 and SH-SY5Y Cells.

    Science.gov (United States)

    Zheng, Nan; Yuan, Peng; Li, Changhao; Wu, Jun; Huang, Jian

    2015-01-01

    Beta-secretase (BACE1) controls an essential step for the generation of amyloid- peptide (Aβ). As Aβ forms the principle pathologies in Alzheimer’s disease, lowering A production by inhibiting BACE1 is a plausible therapeutic approach. In the present study, we identified a natural polyphenol, luteolin, as a potent inhibitor of BACE1 transcription inhuman embryonic kidney 293 (HEK293) and human neuroblastoma (SH-SY5Y) cell lines. Luteolin is capable of suppressing the activation of BACE1 promoter by NF-κB signaling. We further characterized that luteolin interferes with NF-κB signaling by with both directly and indirectly disrupting p65 complex formation. In addition, we discovered that estrogen receptor mediates luteolin’s effect in inhibiting NF-κB signaling inhibiting and BACE1 transcription. Interestingly, the beneficial effects of luteolin may be attributed to selective activation profiles of luteolin to different estrogen receptor subtypes. Our study reports luteolin as a potent BACE1-inhibiting compound, providing useful information in understanding estrogen receptor- and NF-κB-mediated signaling and in regulating BACE1 expression.

  10. Angiotensin receptor-mediated oxidative stress is associated with impaired cardiac redox signaling and mitochondrial function in insulin-resistant rats.

    Science.gov (United States)

    Vázquez-Medina, José Pablo; Popovich, Irina; Thorwald, Max A; Viscarra, Jose A; Rodriguez, Ruben; Sonanez-Organis, Jose G; Lam, Lisa; Peti-Peterdi, Janos; Nakano, Daisuke; Nishiyama, Akira; Ortiz, Rudy M

    2013-08-15

    Activation of angiotensin receptor type 1 (AT1) contributes to NADPH oxidase (Nox)-derived oxidative stress during metabolic syndrome. However, the specific role of AT1 in modulating redox signaling, mitochondrial function, and oxidative stress in the heart remains more elusive. To test the hypothesis that AT1 activation increases oxidative stress while impairing redox signaling and mitochondrial function in the heart during diet-induced insulin resistance in obese animals, Otsuka Long Evans Tokushima Fatty (OLETF) rats (n = 8/group) were treated with the AT1 blocker (ARB) olmesartan for 6 wk. Cardiac Nox2 protein expression increased 40% in OLETF compared with age-matched, lean, strain-control Long Evans Tokushima Otsuka (LETO) rats, while mRNA and protein expression of the H₂O₂-producing Nox4 increased 40-100%. ARB treatment prevented the increase in Nox2 without altering Nox4. ARB treatment also normalized the increased levels of protein and lipid oxidation (nitrotyrosine, 4-hydroxynonenal) and increased the redox-sensitive transcription factor Nrf2 by 30% and the activity of antioxidant enzymes (SOD, catalase, GPx) by 50-70%. Citrate synthase (CS) and succinate dehydrogenase (SDH) activities decreased 60-70%, whereas cardiac succinate levels decreased 35% in OLETF compared with LETO, suggesting that mitochondrial function in the heart is impaired during obesity-induced insulin resistance. ARB treatment normalized CS and SDH activities, as well as succinate levels, while increasing AMPK and normalizing Akt, suggesting that AT1 activation also impairs cellular metabolism in the diabetic heart. These data suggest that the cardiovascular complications associated with metabolic syndrome may result from AT1 receptor-mediated Nox2 activation leading to impaired redox signaling, mitochondrial activity, and dysregulation of cellular metabolism in the heart.

  11. Loss of progesterone receptor-mediated actions induce preterm cellular and structural remodeling of the cervix and premature birth.

    Science.gov (United States)

    Yellon, Steven M; Dobyns, Abigail E; Beck, Hailey L; Kurtzman, James T; Garfield, Robert E; Kirby, Michael A

    2013-01-01

    A decline in serum progesterone or antagonism of progesterone receptor function results in preterm labor and birth. Whether characteristics of premature remodeling of the cervix after antiprogestins or ovariectomy are similar to that at term was the focus of the present study. Groups of pregnant rats were treated with vehicle, a progesterone receptor antagonist (onapristone or mifepristone), or ovariectomized on day 17 postbreeding. As expected, controls given vehicle delivered at term while rats delivered preterm after progesterone receptor antagonist treatment or ovariectomy. Similar to the cervix before term, the preterm cervix of progesterone receptor antagonist-treated rats was characterized by reduced cell nuclei density, decreased collagen content and structure, as well as a greater presence of macrophages per unit area. Thus, loss of nuclear progesterone receptor-mediated actions promoted structural remodeling of the cervix, increased census of resident macrophages, and preterm birth much like that found in the cervix at term. In contrast to the progesterone receptor antagonist-induced advance in characteristics associated with remodeling, ovariectomy-induced loss of systemic progesterone did not affect hypertrophy, extracellular collagen, or macrophage numbers in the cervix. Thus, the structure and macrophage census in the cervix appear sufficient for premature ripening and birth to occur well before term. With progesterone receptors predominantly localized on cells other than macrophages, the findings suggest that interactions between cells may facilitate the loss of progesterone receptor-mediated actions as part of a final common mechanism that remodels the cervix in certain etiologies of preterm and with parturition at term.

  12. Folate-receptor-mediated delivery of InP quantum dots for bioimaging using confocal and two-photon microscopy.

    Science.gov (United States)

    Bharali, Dhruba J; Lucey, Derrick W; Jayakumar, Harishankar; Pudavar, Haridas E; Prasad, Paras N

    2005-08-17

    A novel method for the synthesis of highly monodispersed hydrophillic InP-ZnS nanocrystals and their use as luminescence probes for live cell imaging is reported. Hydrophobic InP-ZnS nanocrystals are prepared by a new method that yields high-quality, luminescent core-shell nanocrystals within 6-8 h of total reaction time. Then by carefully manipulating the surface of these passivated nanocrystals, aqueous dispersions of folate-conjugated nanocrystals (folate-QDs) with high photostability are prepared. By use of confocal microscopy, we demonstrate the receptor-mediated delivery of folic acid conjugated quantum dots into folate-receptor-positive cell lines such as KB cells. These folate-QDs tend to accumulate in multi-vescicular bodies of KB cells after 6 h of incubation. Receptor-mediated delivery was confirmed by comparison with the uptake of these particles in folate-receptor-negative cell lines such as A549. Efficient two-photon excitation of these particles and two-photon imaging using these particles are also demonstrated. The use of these InP-ZnS nanoparticles and their efficient two-photon excitation can be potentially useful for deep tissue imaging for future in vivo studies.

  13. Nicotine alpha 4 beta 2 receptor-mediated free calcium in an animal model of facial nucleus injury

    Institute of Scientific and Technical Information of China (English)

    Dawei Sun; Wenhai Sun; Yanqing Wang; Fugao Zhu; Rui Zhou; Yanjun Wang; Banghua Liu; Xiuming Wan; Huamin Liu

    2010-01-01

    Previous studies have demonstrated that the cholinergic system,via nicotinic receptors,regulates intracellular free calcium levels in the facial nucleus under normal physiological conditions.However,the regulation of nicotinic receptors on free calcium levels following facial nerve injury remains unclear.In the present study,an animal model of facial nerve injury was established,and changes in nicotinic receptor expression following facial nerve injury in rats were detected using reverse transcription polymerase chain reaction.Nicotinic receptor-mediated changes of free calcium levels following facial nucleus injury were determined by laser confocal microscopy.Results showed no significant difference in nicotinic receptor expression between the normal group and the affected facial nerve nucleus.The nicotinic receptor α4β2 subtype increased free calcium levels following facial nerve injury by promoting calcium transmembrane influx,and L-type voltage-gated calcium channel-mediated influx of calcium ions played an important role in promoting calcium transmembrane influx.The nicotinic receptor-mediated increase of free calcium levels following facial nerve injury provides an important mechanism for the repair of facial nerve injury.

  14. H1 and H2 receptors mediate postexercise hyperemia in sedentary and endurance exercise-trained men and women.

    Science.gov (United States)

    McCord, Jennifer L; Halliwill, John R

    2006-12-01

    In sedentary individuals, H(1) receptors mediate the early portion of postexercise skeletal muscle hyperemia, whereas H(2) receptors mediate the later portion. It is not known whether postexercise hyperemia also presents in endurance-trained individuals. We hypothesized that the postexercise skeletal muscle hyperemia would also exist in endurance-trained individuals and that combined blockade of H(1) and H(2) receptors would abolish the long-lasting postexercise hyperemia in trained and sedentary individuals. We studied 28 sedentary and endurance trained men and women before and through 90 min after a 60-min bout of cycling at 60% peak O(2) uptake on control and combined H(1)- and H(2)-receptor antagonist days (fexofenadine and ranitidine). We measured arterial pressure (brachial auscultation) and femoral blood flow (Doppler ultrasound). On the control day, femoral vascular conductance (calculated as flow/pressure) was elevated in all groups 60 min after exercise (sedentary men: Delta86 +/- 35%, trained men, Delta65 +/- 18%; sedentary women, Delta61 +/- 19%, trained women: Delta59 +/- 23%, where Delta is change; all P men: Delta21 +/- 17%, trained men: Delta9 +/- 5%, sedentary women: Delta19 +/- 4%, trained women: Delta11 +/- 11%; all P > 0.16 vs. preexercise; all P men and women. Furthermore, histaminergic mechanisms produce the long-lasting hyperemia in sedentary and endurance-trained individuals.

  15. Receptor-Mediated Endocytosis of Two-Dimensional Nanomaterials Undergoes Flat Vesiculation and Occurs by Revolution and Self-Rotation.

    Science.gov (United States)

    Mao, Jian; Chen, Pengyu; Liang, Junshi; Guo, Ruohai; Yan, Li-Tang

    2016-01-26

    Two-dimensional nanomaterials, such as graphene and transitional metal dichalcogenide nanosheets, are promising materials for the development of antimicrobial surfaces and the nanocarriers for intracellular therapy. Understanding cell interaction with these emerging materials is an urgently important issue to promoting their wide applications. Experimental studies suggest that two-dimensional nanomaterials enter cells mainly through receptor-mediated endocytosis. However, the detailed molecular mechanisms and kinetic pathways of such processes remain unknown. Here, we combine computer simulations and theoretical derivation of the energy within the system to show that the receptor-mediated transport of two-dimensional nanomaterials, such as graphene nanosheet across model lipid membrane, experiences a flat vesiculation event governed by the receptor density and membrane tension. The graphene nanosheet is found to undergo revolution relative to the membrane and, particularly, unique self-rotation around its normal during membrane wrapping. We derive explicit expressions for the formation of the flat vesiculation, which reveals that the flat vesiculation event can be fundamentally dominated by a dimensionless parameter and a defined relationship determined by complicated energy contributions. The mechanism offers an essential understanding on the cellular internalization and cytotoxicity of the emerging two-dimensional nanomaterials.

  16. Cell Type-Specific Delivery of RNAi by Ligand-Functionalized Curdlan Nanoparticles: Balancing the Receptor Mediation and the Charge Motivation.

    Science.gov (United States)

    Wu, Yinga; Cai, Jia; Han, Jingfen; Baigude, Huricha

    2015-09-30

    Tissue-specific delivery of therapeutic RNAi has great potential for clinical applications. Receptor-mediated endocytosis plays a crucial role in targeted delivery of biotherapeutics including short interfering RNA (siRNA). Previously we reported a novel Curdlan-based nanoparticle for intracellular delivery of siRNA. Here we designed a nanoparticle based on ligand-functionalized Curdlan. Disaccharides were site-specifically conjugated to 6-deoxy-6-amino Curdlan, and the cell line specificity, cellular uptake, cytotoxicity, and siRNA delivery efficiency of the corresponding disaccharide-modified 6-deoxy-6-amino-Curdlan were investigated. Observation by fluorescence microscopy as well as flow cytometry showed that galactose-containing Curdlan derivatives delivered fluorescently labeled short nucleic acid to HepG2 cells expressing ASGPR receptor but not in other cells lacking surface ASGPR protein. Moreover, highly galactose-substituted Curdlan derivatives delivered siRNA specifically to ASGPR-expressing cells and induced RNAi activities, silencing endogenous GAPDH gene expression. Our data demonstrated that galactose-functionalized 6-deoxy-6-amino-Curdlan is a promising carrier for short therapeutic nucleic acids for clinical applications.

  17. Metabolism of glycosylated human salivary amylase: in vivo plasma clearance by rat hepatic endothelial cells and in vitro receptor mediated pinocytosis by rat macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Niesen, T.E.; Alpers, D.H.; Stahl, P.D.; Rosenblum, J.L.

    1984-09-01

    Salivary-type amylase normally comprises about 60% of the amylase activity in human serum, but only a small fraction is a glycosylated isoenzyme (amylase A). In contrast, 1/3 of amylase in human saliva is glycosylated. Since glycosylation can affect circulatory clearance, we studied the clearance of amylase A in rats and its uptake by rat alveolar macrophages. Following intravenous injection, /sup 125/I-labeled amylase A disappeared rapidly from plasma (t 1/2 . 9 min) and accumulated in the liver. Simultaneous injection of mannose-albumin slowed its clearance to a rate comparable to that of /sup 125/I-labeled nonglycosylated salivary amylase (t 1/2 . 45 min). In contrast, galactose-albumin had no effect. Electron microscope autoradiography of the liver following injection of /sup 125/I-labeled amylase A revealed a localization of grains over the hepatic endothelial cells. In vitro studies indicated that amylase A is taken up by alveolar macrophages via receptor-mediated pinocytosis. Uptake was linear over time, saturable, and inhibited by mannan and mannose-albumin, but not by galactose-albumin. We conclude that amylase A, which is a naturally occurring human glycoprotein with at most three terminal L-fucose residues per molecule, is recognized in rats by a mannose receptor located on hepatic endothelial cells. We speculate that this receptor, by rapidly clearing circulating amylase A, may be responsible for the low level of amylase A in human serum.

  18. Optogenetic Evocation of Field Inhibitory Postsynaptic Potentials in Hippocampal Slices: A Simple and Reliable Approach for Studying Pharmacological Effects on GABAA and GABAB Receptor-Mediated Neurotransmission

    Directory of Open Access Journals (Sweden)

    Julien eDine

    2014-01-01

    Full Text Available The GABAergic system is the main source of inhibition in the mammalian brain. Consequently, much effort is still made to develop new modulators of GABAergic synaptic transmission. In contrast to glutamatergic postsynaptic potentials (PSPs, accurate monitoring of GABA receptor-mediated PSPs (GABAR-PSPs and their pharmacological modulation in brain tissue invariably requires the use of intracellular recording techniques. However, these techniques are expensive, time- and labor-consuming, and, in case of the frequently employed whole-cell patch-clamp configuration, impact on intracellular ion concentrations, signaling cascades, and pH buffering systems. Here, we describe a novel approach to circumvent these drawbacks. In particular, we demonstrate in mouse hippocampal slices that selective optogenetic activation of interneurons leads to prominent field inhibitory GABAAR- and GABABR-PSPs in area CA1 which are easily and reliably detectable by a single extracellular recording electrode. The field PSPs exhibit typical temporal and pharmacological characteristics, display pronounced paired-pulse depression, and remain stable over many consecutive evocations. Additionally validating the methodological value of this approach, we further show that the neuroactive steroid 5-THDOC (5 µM shifts the inhibitory GABAAR-PSPs towards excitatory ones.

  19. The role of G protein coupled receptor-mediated signaling in the biological properties of Acanthamoeba castellanii of the T4 genotype.

    Science.gov (United States)

    Aqeel, Yousuf; Siddiqui, Ruqaiyyah; Manan, Zainab; Khan, Naveed Ahmed

    2015-04-01

    Despite advances in antimicrobial chemotherapy and supportive care, the prognosis of Acanthamoeba infections remains poor, suggesting that new targets are needed that can affect parasite survival and host-pathogen interactions. G proteins and their coupled receptors are well known regulators of a variety of cellular functions. The overall aim of the present study was to study the role of G-protein coupled receptor, β adrenergic receptor on the biology and pathogenesis of keratitis isolate of Acanthamoeba castellanii of the T4 genotype. Inhibition of β adrenergic receptor using antagonist, propranolol had detrimental effects on the extracellular proteolytic activities A. castellanii as determined using zymographic assays. Conversely, β adrenergic receptor agonist, isoprenaline showed increased proteases. Interestingly, β adrenergic receptor inhibition affected A. castellanii growth (using amoebistatic assays), viability (using amoebicidal assays by measuring uptake of Trypan blue) and encystation as determined by trophozoite transformation into the cyst form. Pre-treatment of parasites with propranolol hampered A. castellanii-mediated human brain microvascular endothelial cell cytotoxicity, as measured by the lacatate dehydrogenase release. The aforementioned findings suggest that G-protein coupled receptor, β adrenergic receptor-mediated signaling in A. castellanii biology and pathogenesis may offer new pharmacological targets.

  20. Mechanical stress triggers cardiomyocyte autophagy through angiotensin II type 1 receptor-mediated p38MAP kinase independently of angiotensin II.

    Directory of Open Access Journals (Sweden)

    Li Lin

    Full Text Available Angiotensin II (Ang II type 1 (AT1 receptor is known to mediate a variety of physiological actions of Ang II including autophagy. However, the role of AT1 receptor in cardiomyocyte autophagy triggered by mechanical stress still remains elusive. The aim of this study was therefore to examine whether and how AT1 receptor participates in cardiomyocyte autophagy induced by mechanical stresses. A 48-hour mechanical stretch and a 4-week transverse aorta constriction (TAC were imposed to cultured cardiomyocytes of neonatal rats and adult male C57B/L6 mice, respectively, to induce cardiomyocyte hypertrophy prior to the assessment of cardiomyocyte autophagy using LC3b-II. Losartan, an AT1 receptor blocker, but not PD123319, the AT2 inhibitor, was found to significantly reduce mechanical stretch-induced LC3b-II upregulation. Moreover, inhibition of p38MAP kinase attenuated not only mechanical stretch-induced cardiomyocyte hypertrophy but also autophagy. To the contrary, inhibition of ERK and JNK suppressed cardiac hypertrophy but not autophagy. Intriguingly, mechanical stretch-induced autophagy was significantly inhibited by Losartan in the absence of Ang II. Taken together, our results indicate that mechanical stress triggers cardiomyocyte autophagy through AT1 receptor-mediated activation of p38MAP kinase independently of Ang II.

  1. Permanent Distal Occlusion of Middle Cerebral Artery in Rat Causes Local Increased ETB, 5-HT1B and AT1 Receptor-Mediated Contractility Downstream of Occlusion

    DEFF Research Database (Denmark)

    Rasmussen, Marianne N P; Hornbak, Malene; Larsen, Stine S;

    2013-01-01

    a model of permanent distal occlusion of rat middle cerebral arteries, we investigated whether there was a regional difference in receptor-mediated contractility of segments located upstream and downstream of the occlusion site. The contractile response to endothelin, angiotensin and 5-hydroxytryptamine...... occlusion without significant visible infarct resulted in locally increased ETB, angiotensin type 1 and 5-hydroxytryptamine 1B receptor-mediated contractile responses only in segments located downstream of the occlusion site. This suggests lack of wall stress as an initiating trigger leading to regulation...

  2. Genetically designed biomolecular capping system for mesoporous silica nanoparticles enables receptor-mediated cell uptake and controlled drug release

    CERN Document Server

    Datz, Stefan; Gattner, Michael; Weiss, Veronika; Brunner, Korbinian; Bretzler, Johanna; von Schirnding, Constantin; Spada, Fabio; Engelke, Hanna; Vrabel, Milan; Bräuchle, Christoph; Carell, Thomas; Bein, Thomas

    2015-01-01

    Effective and controlled drug delivery systems with on-demand release and targeting abilities have received enormous attention for biomedical applications. Here, we describe a novel enzyme-based cap system for mesoporous silica nanoparticles (MSNs) that is directly combined with a targeting ligand via bio-orthogonal click chemistry. The capping system is based on the pH-responsive binding of an aryl-sulfonamide-functionalized MSN and the enzyme carbonic anhydrase (CA). An unnatural amino acid (UAA) containing a norbornene moiety was genetically incorporated into CA. This UAA allowed for the site-specific bio-orthogonal attachment of even very sensitive targeting ligands such as folic acid and anandamide. This leads to specific receptor-mediated cell and stem cell uptake. We demonstrate the successful delivery and release of the chemotherapeutic agent Actinomycin D to KB cells. This novel nanocarrier concept provides a promising platform for the development of precisely controllable and highly modular theranos...

  3. Cholera Toxin Inhibits the T-Cell Antigen Receptor-Mediated Increases in Inositol Trisphosphate and Cytoplasmic Free Calcium

    Science.gov (United States)

    Imboden, John B.; Shoback, Dolores M.; Pattison, Gregory; Stobo, John D.

    1986-08-01

    The addition of monoclonal antibodies to the antigen receptor complex on the malignant human T-cell line Jurkat generates increases in inositol trisphosphate and in the concentration of cytoplasmic free calcium. Exposure of Jurkat cells to cholera toxin for 3 hr inhibited these receptor-mediated events and led to a selective, partial loss of the antigen receptor complex from the cellular surface. None of the effects of cholera toxin on the antigen receptor complex were mimicked by the B subunit of cholera toxin or by increasing intracellular cAMP levels with either forskolin or 8-bromo cAMP. These results suggest that a cholera toxin substrate can regulate signal transduction by the T-cell antigen receptor.

  4. γ-Aminobutyric Acid B Receptor Mediated Inhibition of Gonadotropin-Releasing Hormone Neurons Is Suppressed by Kisspeptin-G Protein-Coupled Receptor 54 Signaling

    Science.gov (United States)

    Zhang, Chunguang; Bosch, Martha A.; Rønnekleiv, Oline K.; Kelly, Martin J.

    2009-01-01

    γ-Aminobutyric acid (GABA) is one of the most important neurotransmitters that regulate the excitability of GnRH neurons. Numerous studies have shown that GABA activates Cl− currents in GnRH neurons, and these effects are antagonized by GABAA receptor antagonists. The GABAB receptor is a heterodimer composed of GABAB R1 and R2, and although both subunits have been localized in GnRH neurons, nothing is known about the cellular signaling of this Gαi,o-coupled receptor in GnRH neurons. Using whole-cell recordings from mouse enhanced green fluorescent protein-GnRH neurons, we found that the GABAB receptor agonist baclofen hyperpolarized GnRH neurons through activation of an inwardly rectifying K+ current in a concentration-dependent manner. The effects of baclofen were antagonized by the selective GABAB receptor antagonist CGP 52432 with a Ki (inhibitory constant) of 85 nm. Furthermore, in the presence of the GABAA receptor antagonist picrotoxin, GABA hyperpolarized GnRH neurons in a similar manner. Treatment with 17β-estradiol as compared with oil vehicle did not significantly alter either the EC50 for the baclofen-induced response (0.8 ± 0.1 vs. 1.0 ± 0.1 μm, respectively) or the maximal outward current (10.8 ± 1.7 pA vs. 11.4 ± 0.6 pA, respectively) in GnRH neurons. However, the outward current (and membrane hyperpolarization) was abrogated by submaximal concentrations of the G protein-coupled receptor 54 (GPR54) agonist kisspeptin-10 in both groups, indicating that Gαq-coupled (GPR54) can desensitize the GABAB receptor-mediated response. Therefore, the activation of GABAB receptors in GnRH neurons may provide increased inhibitory tone during estrogen-negative feedback states that is attenuated by kisspeptin during positive feedback. PMID:19164470

  5. Elevated potassium elicits recurrent surges of large GABAA-receptor-mediated post-synaptic currents in hippocampal CA3 pyramidal neurons.

    Science.gov (United States)

    Shin, Damian Seung-Ho; Yu, Wilson; Sutton, Alex; Calos, Megan; Carlen, Peter Louis

    2011-03-01

    Previously, we found that rat hippocampal CA3 interneurons become hyperactive with increasing concentrations of extracellular K(+) up to 10 mM. However, it is unclear how this enhanced interneuronal activity affects pyramidal neurons. Here we voltage-clamped rat hippocampal CA3 pyramidal neurons in vitro at 0 mV to isolate γ-aminobutyric acid (GABA)-activated inhibitory post-synaptic currents (IPSCs) and measured these in artificial cerebrospinal fluid (aCSF) and with 10 mM K(+) bath perfusion. In aCSF, small IPSCs were present with amplitudes of 0.053 ± 0.007 nA and a frequency of 0.27 ± 0.14 Hz. With 10 mM K(+) perfusion, IPSCs increased greatly in frequency and amplitude, culminating in surge events with peak amplitudes of 0.56 ± 0.08 nA, that appeared and disappeared cyclically with durations lasting 2.02 ± 0.37 min repeatedly, up to 10 times over a 30-min bath perfusion of elevated K(+). These large IPSCs were GABA(A)-receptor mediated and did not involve significant desensitization of this receptor. Perfusion of a GABA transporter inhibitor (NO-711), glutamate receptor inhibitors CNQX and APV, or a gap junctional blocker (carbenoxolone) prevented the resurgence of large IPSCs. Pressure ejected sucrose resulted in the abolishment of subsequent surges. No elevated K(+)-mediated surges were observed in CA3 interneurons from the stratum oriens layer. In conclusion, these cyclic large IPSC events observable in CA3 pyramidal neurons in 10 mM KCl may be due to transient GABA depletion from continuously active interneuronal afferents.

  6. Loss of progesterone receptor-mediated actions induce preterm cellular and structural remodeling of the cervix and premature birth.

    Directory of Open Access Journals (Sweden)

    Steven M Yellon

    Full Text Available A decline in serum progesterone or antagonism of progesterone receptor function results in preterm labor and birth. Whether characteristics of premature remodeling of the cervix after antiprogestins or ovariectomy are similar to that at term was the focus of the present study. Groups of pregnant rats were treated with vehicle, a progesterone receptor antagonist (onapristone or mifepristone, or ovariectomized on day 17 postbreeding. As expected, controls given vehicle delivered at term while rats delivered preterm after progesterone receptor antagonist treatment or ovariectomy. Similar to the cervix before term, the preterm cervix of progesterone receptor antagonist-treated rats was characterized by reduced cell nuclei density, decreased collagen content and structure, as well as a greater presence of macrophages per unit area. Thus, loss of nuclear progesterone receptor-mediated actions promoted structural remodeling of the cervix, increased census of resident macrophages, and preterm birth much like that found in the cervix at term. In contrast to the progesterone receptor antagonist-induced advance in characteristics associated with remodeling, ovariectomy-induced loss of systemic progesterone did not affect hypertrophy, extracellular collagen, or macrophage numbers in the cervix. Thus, the structure and macrophage census in the cervix appear sufficient for premature ripening and birth to occur well before term. With progesterone receptors predominantly localized on cells other than macrophages, the findings suggest that interactions between cells may facilitate the loss of progesterone receptor-mediated actions as part of a final common mechanism that remodels the cervix in certain etiologies of preterm and with parturition at term.

  7. Characterization of GABA/sub A/ receptor-mediated /sup 36/chloride uptake in rat brain synaptoneurosomes

    Energy Technology Data Exchange (ETDEWEB)

    Luu, M.D.; Morrow, A.L.; Paul, S.M.; Schwartz, R.D.

    1987-09-07

    ..gamma..-Aminobutyric acid (GABA) receptor-mediated /sup 36/chloride (/sup 36/Cl/sup -/) uptake was measured in synaptoneurosomes from rat brain. GABA and GABA agonists stimulated /sup 36/Cl/sup -/ uptake in a concentration-dependent manner with the following order of potency: Muscimol>GABA>piperidine-4-sulfonic acid (P4S)>4,5,6,7-tetrahydroisoxazolo-(5,4-c)pyridin-3-ol (THIP)=3-aminopropanesulfonic acid (3APS)>>taurine. Both P4S and 3APS behaved as partial agonists, while the GABA/sub B/ agonist, baclofen, was ineffective. The response to muscimol was inhibited by bicuculline and picrotoxin in a mixed competitive/non-competitive manner. Other inhibitors of GABA receptor-opened channels or non-neuronal anion channels such as penicillin, picrate, furosemide and disulfonic acid stilbenes also inhibited the response to muscimol. A regional variation in muscimol-stimulated /sup 36/Cl/sup -/ uptake was observed; the largest responses were observed in the cerebral cortex, cerebellum and hippocampus, moderate responses were obtained in the striatum and hypothalamus and the smallest response was observed in the pons-medulla. GABA receptor-mediated /sup 36/Cl/sup -/ uptake was also dependent on the anion present in the media. The muscinol response varied in media containing the following anions: Br/sup -/>Cl/sup -/greater than or equal toNO/sub 3//sup -/>I/sup -/greater than or equal toSCN/sup -/>>C/sub 3/H/sub 5/OO/sup -/greater than or equal toClO/sub 4//sup -/>F/sup -/, consistent with the relative anion permeability through GABA receptor-gated anion channels and the enhancement of convulsant binding to the GABA receptor-gated Cl/sup -/ channel. 43 references, 4 figures, 3 tables.

  8. Probes for Narcotic Receptor Mediated Phenomena. 39. Enantiomeric N-Substituted Benzofuro[2,3-c]pyridin-6-ols: Synthesis and Topological Relationship to Oxide-Bridged Phenylmorphans

    Science.gov (United States)

    2009-01-01

    1989, 32, 2221–2226. (4) Burke, T. R. Jr.; Jacobson, A. E.; Rice, K. C.; Silverton , J. V. Probes for Narcotic Receptor Mediated Phenomena. 4...Chem. 1984, 49, 1051–1056. (5) Burke, T. R.Jr.; Jacobson, A. E.; Rice, K. C.; Silverton , J. V. Probes for Narcotic Receptor Mediated Phenomena. 6

  9. H3 receptor-mediated inhibition of noradrenaline release: an investigation into the involvement of Ca2+ and K+ ions, G protein and adenylate cyclase.

    Science.gov (United States)

    Schlicker, E; Kathmann, M; Detzner, M; Exner, H J; Göthert, M

    1994-07-01

    The present study was aimed at the identification of mechanisms following the activation of histamine H3 receptors. Mouse brain cortex slices preincubated with 3H-noradrenaline were superfused and the (H3 receptor-mediated) effect of histamine on the electrically evoked tritium overflow was studied under a variety of conditions. The extent of inhibition produced by histamine was inversely related to the frequency of stimulation used to evoke tritium overflow and to the Ca2+ concentration in the superfusion medium. An activator (levcromakalim) and blocker (glibenclamide) of ATP-dependent K+ channels did not affect the electrically evoked tritium overflow and its inhibition by histamine. A blocker of voltage-sensitive K+ channels, tetraethylammonium (TEA), increased the evoked overflow and attenuated the inhibitory effect of histamine. TEA also reduced the inhibitory effect of noradrenaline and prostaglandin E2 on the evoked overflow. When the facilitatory effect of TEA on the evoked overflow was compensated for by reducing the Ca2+ concentration in the superfusion medium, TEA did no longer attenuate the effect of histamine. Exposure of the slices to the SH group-alkylating agent N-ethylmaleimide increased the evoked overflow and attenuated the inhibitory effect of histamine; both effects were counteracted by the SH group-protecting agent dithiothreitol, which, by itself, did not affect the evoked overflow and its inhibition by histamine. Mouse brain cortex membranes were used to study the effect of the H3 receptor agonist R-(-)-alpha-methylhistamine on the basal cAMP accumulation and on the accumulation stimulated by forskolin or noradrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. ERK/Egr-1 signaling pathway is involved in CysLT2 receptor-mediated IL-8 production in HEK293 cells.

    Science.gov (United States)

    Lin, Kana; Fang, Sanhua; Cai, Beilei; Huang, Xueqin; Zhang, Xiayan; Lu, Yunbi; Zhang, Weiping; Wei, Erqing

    2014-07-01

    The CysLT2 receptor is involved in myocardial ischemia/reperfusion injury, differentiation of colorectal cancers, bleomycin-induced pulmonary inflammation and fibrosis. However, the signal transduction of cysteinyl leukotriene receptor 2 (CysLT2) in inflammatory responses remains to be clarified. In HEK293 cells stably expressing hCysLT1, hCysLT2 and rGPR17, we determined the signaling pathways for interleukin-8 (IL-8) production after CysLT2 receptor activation. HEK293 cells were stably transfected with the recombinant plasmids of pcDNA3.1(+)-hCysLT1, pcDNA3.1(+)-hCysLT2 and pcDNA3.1-rGPR17. Leukotriene C4 (LTC4) and LTD4 were used as the agonists to induce IL-8 production and the related changes in signal molecules. We found that LTC4 and LTD4 significantly induced IL-8 promoter activation in the HEK293 cells stably expressing hCysLT2, but not in those expressing hCysLT1 and rGPR17. In hCysLT2-HEK293 cells, LTC4 induced elevation of intracellular calcium, ERK1/2 phosphorylation and Egr-1 expression, and stimulated IL-8 expression and release. These responses were blocked by the selective CysLT2 receptor antagonist HAMI3379. The ERK1/2 inhibitor U0126 inhibited Egr-1 and IL-8 expression as well as IL-8 release, but the JNK and p38 inhibitors did not have the inhibitory effects. Down-regulation of Egr-1 by RNA interference with its siRNA inhibited the LTC4-induced IL-8 expression and release. In conclusion, these findings indicate the ERK-Egr-1 pathway of CysLT2 receptors mediates IL-8 production induced by the pro-inflammatory mediators LTC4 and LTD4.

  11. Development of a stably transfected estrogen receptor-mediated luciferase reporter gene assay in the human T47D breast cancer cell line.

    Science.gov (United States)

    Legler, J; van den Brink, C E; Brouwer, A; Murk, A J; van der Saag, P T; Vethaak, A D; van der Burg, B

    1999-03-01

    Development of an estrogen receptor-mediated, chemical-activated luciferase reporter gene-expression (ER-CALUX) assay was attempted by stable transfection of luciferase reporter genes in a number of cell lines. Stable transfection of the chimeric Gal4 estrogen receptor and luciferase gene constructs in MCF-7 breast cancer and Hepa.1c1c7 mouse hepatoma cell lines, as well as transfection of a newly constructed luciferase reporter gene pEREtata-Luc in the ECC-1 human endometrial cell line, resulted in constitutive, non-estradiol-inducible clones. Stable transfection of pEREtata-Luc in the T47D breast cancer cell line, however, resulted in an extremely sensitive, highly responsive cell line. Following a 24-h exposure to estradiol (E2), stably transfected T47D.Luc cells demonstrated a detection limit of 0.5 pM, an EC50 of 6 pM, and a maximum induction of 100-fold relative to solvent controls. No clear reduction in responsiveness has been found over extended culture periods (50 passages). Anti-estrogens ICI 182,780, TCDD, and tamoxifen inhibited the estradiol-mediated luciferase induction. Genistein, nonylphenol, and o,p'DDT were the most potent (pseudo-)estrogens tested in this system (EC50 100, 260, and 660 nM, respectively). Determination of interactive effects of the (pseudo-)estrogens nonylphenol, o,p'DDT, chlordane, endosulfan, dieldrin, and methoxychlor revealed that, in combination with 3 pM E2, (pseudo-)estrogens were additive. Slightly more than additive effects (less than 2-fold) were found for combinations of dieldrin and endosulfan tested in the range of 3 to 6 microM. At these concentrations, the combination of endosulfan and chlordane demonstrated additive interaction. The ER-CALUX assay with T47D cells can provide a sensitive, responsive, and rapid in vitro system to detect and measure substances with potential (anti-)estrogenic activity.

  12. A Boolean Network Model of Nuclear Receptor Mediated Cell Cycle Progression (S)

    Science.gov (United States)

    Nuclear receptors (NRs) are ligand-activated transcription factors that regulate a broad range of cellular processes. Hormones, lipids and xenobiotics have been shown to activate NRs with a range of consequences on development, metabolism, oxidative stress, apoptosis, and prolif...

  13. A Boolean Network Model of Nuclear Receptor Mediated Cell Cycle Progression

    Science.gov (United States)

    Nuclear receptors (NRs) are ligand-activated transcription factors that regulate a broad range of cellular processes. Hormones, lipids and xenobiotics have been shown to activate NRs with a range of consequences on development, metabolism, oxidative stress, apoptosis, and prolif...

  14. Phenobarbital but not diazepam reduces AMPA/Kainate receptor mediated currents and exerts opposite actions on initial seizures in the neonatal rat hippocampus

    Directory of Open Access Journals (Sweden)

    Romain eNardou

    2011-07-01

    Full Text Available Diazepam (DZP and phenobarbital (PB are extensively used as first and second line drugs to treat acute seizures in neonates and their actions are thought to be mediated by increasing the actions of GABAergic signals. Yet, their efficacy is variable with occasional failure or even aggravation of recurrent seizures questioning whether other mechanisms are not involved in their actions. We have now compared the effects of DZP and PB on ictal-like events (ILEs in an in vitro model of mirror focus (MF. Using the three-compartment chamber with the two immature hippocampi and their commissural fibers placed in 3 different compartments, kainate was applied to one hippocampus and PB or DZP to the contralateral one, either after one ILE or after many recurrent ILEs that produce an epileptogenic MF. We report that in contrast to PB, DZP aggravated propagating ILEs from the start and did not prevent the formation of MF. PB reduced and DZP increased the network driven Giant Depolarising Potentials suggesting that PB may exert additional actions that are not mediated by GABA signalling. In keeping with this, PB but not DZP reduced field potentials recorded in the presence of GABA and NMDA receptor antagonists. These effects are mediated by a direct action on AMPA/Kainate receptors since PB: i reduced AMPA/Kainate receptor mediated currents induced by focal applications of glutamate ; ii reduced the amplitude and the frequency of AMPA but not NMDA receptor mediated miniature EPSCs; iii augmented the number of AMPA receptor mediated EPSCs failures evoked by minimal stimulation. These effects persisted in MF. Therefore, PB exerts its anticonvulsive actions partly by reducing AMPA/Kainate receptors mediated EPSCs in addition to the pro-GABA effects. We suggest that PB may have advantage over DZP in the treatment of initial neonatal seizures since the additional reduction of glutamate receptors mediated signals may reduce the severity of neonatal seizures.

  15. Mas receptor mediates cardioprotection of angiotensin-(1-7) against Angiotensin II-induced cardiomyocyte autophagy and cardiac remodelling through inhibition of oxidative stress.

    Science.gov (United States)

    Lin, Li; Liu, Xuebo; Xu, Jianfeng; Weng, Liqing; Ren, Jun; Ge, Junbo; Zou, Yunzeng

    2016-01-01

    Angiotensin II (Ang II) plays an important role in the onset and development of cardiac remodelling associated with changes of autophagy. Angiotensin1-7 [Ang-(1-7)] is a newly established bioactive peptide of renin-angiotensin system, which has been shown to counteract the deleterious effects of Ang II. However, the precise impact of Ang-(1-7) on Ang II-induced cardiomyocyte autophagy remained essentially elusive. The aim of the present study was to examine if Ang-(1-7) inhibits Ang II-induced autophagy and the underlying mechanism involved. Cultured neonatal rat cardiomyocytes were exposed to Ang II for 48 hrs while mice were infused with Ang II for 4 weeks to induce models of cardiac hypertrophy in vitro and in vivo. LC3b-II and p62, markers of autophagy, expression were significantly elevated in cardiomyocytes, suggesting the presence of autophagy accompanying cardiac hypertrophy in response to Ang II treatment. Besides, Ang II induced oxidative stress, manifesting as an increase in malondialdehyde production and a decrease in superoxide dismutase activity. Ang-(1-7) significantly retarded hypertrophy, autophagy and oxidative stress in the heart. Furthermore, a role of Mas receptor in Ang-(1-7)-mediated action was assessed using A779 peptide, a selective Mas receptor antagonist. The beneficial responses of Ang-(1-7) on cardiac remodelling, autophagy and oxidative stress were mitigated by A779. Taken together, these result indicated that Mas receptor mediates cardioprotection of angiotensin-(1-7) against Ang II-induced cardiomyocyte autophagy and cardiac remodelling through inhibition of oxidative stress. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  16. Does ligand-receptor mediated competitive effect or penetrating effect of iRGD peptide when co-administration with iRGD-modified SSL?

    Science.gov (United States)

    Zhang, Wei-Qiang; Yu, Ke-Fu; Zhong, Ting; Luo, Li-Min; Du, Ruo; Ren, Wei; Huang, Dan; Song, Ping; Li, Dan; Zhao, Yang; Wang, Chao; Zhang, Xuan

    2015-12-01

    Ligand-mediated targeting of anticancer therapeutic agents is a useful strategy for improving anti-tumor efficacy. It has been reported that co-administration of a tumor-penetrating peptide iRGD (CRGDK/RGPD/EC) enhances the efficacy of anticancer drugs. Here, we designed an experiment involving co-administration of iRGD-SSL-DOX with free iRGD to B16-F10 tumor bearing mice to examine the action of free iRGD. We also designed an experiment to investigate the location of iRGD-modified SSL when co-administered with free iRGD or free RGD to B16-F10 tumor bearing nude mice. Considering the sequence of iRGD, we selected the GPDC, RGD and CRGDK as targeting ligands to investigate the targeting effect of these peptides compared with iRGD on B16-F10 and MCF-7 cells, with or without enzymatic degradation. Finally, we selected free RGD, free CRGDK and free iRGD as ligand to investigate the inhibitory effect on RGD-, CRGDK- or iRGD-modified SSL on B16-F10 or MCF-7 cells. Our results indicated that iRGD targeting to tumor cells was ligand-receptor mediated involving RGD to αv-integrin receptor and CRGDK to NRP-1 receptor. Being competitive effect, the administration of free iRGD would not be able to further enhance the anti-tumor activity of iRGD-modified SSL. There is no need to co-administrate of free iRGD with the iRGD-modified nanoparticles for further therapeutic benefit.

  17. Phosphorylation of the ryanodine receptor mediates the cardiac fight or flight response in mice

    National Research Council Canada - National Science Library

    Shan, Jian; Kushnir, Alexander; Betzenhauser, Matthew J; Reiken, Steven; Li, Jingdong; Lehnart, Stephan E; Lindegger, Nicolas; Mongillo, Marco; Mohler, Peter J; Marks, Andrew R

    2010-01-01

    During the classic "fight-or-flight" stress response, sympathetic nervous system activation leads to catecholamine release, which increases heart rate and contractility, resulting in enhanced cardiac output...

  18. Glucocorticoid receptor-mediated induction of glutamine synthetase in skeletal muscle cells in vitro

    Science.gov (United States)

    Max, Stephen R.; Thomas, John W.; Banner, Carl; Vitkovic, Ljubisa; Konagaya, Masaaki

    1987-01-01

    The regulation by glucocorticoids of glutamine synthetase in L6 muscle cells in culture is studied. Glutamine synthetase activity was strikingly enhanced by dexamethasone. The dexamethasone-mediated induction of glutamine synthetase activity was blocked by RU38486, a glucocorticoid antagonist, indicating the involvement of intracellular glucocorticoid receptors in the induction process. RU38486 alone was without effect. Northern blot analysis revealed that dexamethasone-mediated enhancement of glutamine synthetase activity involves increased levels of glutamine synthetase mRNA. Glucocorticoids regulate the expression of glutamine synthetase mRNA in cultured muscle cells via interaction with intracellular receptors. Such regulation may be relevant to control of glutamine production by muscle.

  19. Genetically designed biomolecular capping system for mesoporous silica nanoparticles enables receptor-mediated cell uptake and controlled drug release

    Science.gov (United States)

    Datz, Stefan; Argyo, Christian; Gattner, Michael; Weiss, Veronika; Brunner, Korbinian; Bretzler, Johanna; von Schirnding, Constantin; Torrano, Adriano A.; Spada, Fabio; Vrabel, Milan; Engelke, Hanna; Bräuchle, Christoph; Carell, Thomas; Bein, Thomas

    2016-04-01

    Effective and controlled drug delivery systems with on-demand release and targeting abilities have received enormous attention for biomedical applications. Here, we describe a novel enzyme-based cap system for mesoporous silica nanoparticles (MSNs) that is directly combined with a targeting ligand via bio-orthogonal click chemistry. The capping system is based on the pH-responsive binding of an aryl-sulfonamide-functionalized MSN and the enzyme carbonic anhydrase (CA). An unnatural amino acid (UAA) containing a norbornene moiety was genetically incorporated into CA. This UAA allowed for the site-specific bio-orthogonal attachment of even very sensitive targeting ligands such as folic acid and anandamide. This leads to specific receptor-mediated cell and stem cell uptake. We demonstrate the successful delivery and release of the chemotherapeutic agent Actinomycin D to KB cells. This novel nanocarrier concept provides a promising platform for the development of precisely controllable and highly modular theranostic systems.Effective and controlled drug delivery systems with on-demand release and targeting abilities have received enormous attention for biomedical applications. Here, we describe a novel enzyme-based cap system for mesoporous silica nanoparticles (MSNs) that is directly combined with a targeting ligand via bio-orthogonal click chemistry. The capping system is based on the pH-responsive binding of an aryl-sulfonamide-functionalized MSN and the enzyme carbonic anhydrase (CA). An unnatural amino acid (UAA) containing a norbornene moiety was genetically incorporated into CA. This UAA allowed for the site-specific bio-orthogonal attachment of even very sensitive targeting ligands such as folic acid and anandamide. This leads to specific receptor-mediated cell and stem cell uptake. We demonstrate the successful delivery and release of the chemotherapeutic agent Actinomycin D to KB cells. This novel nanocarrier concept provides a promising platform for the

  20. A3 Adenosine receptors mediate oligodendrocyte death and ischemic damage to optic nerve.

    Science.gov (United States)

    González-Fernández, Estíbaliz; Sánchez-Gómez, María Victoria; Pérez-Samartín, Alberto; Arellano, Rogelio O; Matute, Carlos

    2014-02-01

    Adenosine receptor activation is involved in myelination and in apoptotic pathways linked to neurodegenerative diseases. In this study, we investigated the effects of adenosine receptor activation in the viability of oligodendrocytes of the rat optic nerve. Selective activation of A3 receptors in pure cultures of oligodendrocytes caused concentration-dependent apoptotic and necrotic death which was preceded by oxidative stress and mitochondrial membrane depolarization. Oligodendrocyte apoptosis induced by A3 receptor activation was caspase-dependent and caspase-independent. In addition to dissociated cultures, incubation of optic nerves ex vivo with adenosine and the A3 receptor agonist 2-CI-IB-MECA(1-[2-Chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-b-D-ribofuranuronamide)-induced caspase-3 activation, oligodendrocyte damage, and myelin loss, effects which were prevented by the presence of caffeine and the A3 receptor antagonist MRS 1220 (N-[9-Chloro-2-(2-furanyl)[1,2,4]-triazolo [1,5-c]quinazolin-5-yl]benzene acetamide). Finally, ischemia-induced injury and functional loss to the optic nerve was attenuated by blocking A3 receptors. Together, these results indicate that adenosine may trigger oligodendrocyte death via activation of A3 receptors and suggest that this mechanism contributes to optic nerve and white matter ischemic damage.

  1. Berberine reduces Toll-like receptor-mediated macrophage migration by suppression of Src enhancement.

    Science.gov (United States)

    Cheng, Wei-Erh; Ying Chang, Miao; Wei, Jyun-Yan; Chen, Yen-Jen; Maa, Ming-Chei; Leu, Tzeng-Horng

    2015-06-15

    Berberine is an isoquinoline with anti-inflammatory activity. We previously demonstrated that there was a loop of signal amplification between nuclear factor kappa B and Src for macrophage mobility triggered by the engagement of Toll-like receptors (TLRs). The simultaneous suppression of lipopolysaccharide (LPS)-mediated upregulation of inducible nitric oxide synthase, cyclooxygenase 2, and cell mobility in berberine-treated macrophages suggested Src might be a target of berberine. Indeed, th reduced migration, greatly suppressed Src induction in both protein and RNA transcript by berberine were observed in macrophages exposed to LPS, peptidoglycan, polyinosinic-polycytidylic acid, and CpG-oligodeoxynucleotides. In addition to Src induction, berberine also inhibited LPS-mediated Src activation in Src overexpressing macrophages and S-nitroso-N-acetylpenicillamine (a nitric oxide donor) could partly restore it. Moreover, berberine suppressed Src activity in fibronectin-stimulated macrophages and in v-Src transformed cells. These results implied that by effectively reducing Src expression and activity, berberine inhibited TLR-mediated cell motility in macrophages.

  2. Bladder cancer cell growth and motility implicate cannabinoid 2 receptor-mediated modifications of sphingolipids metabolism

    Science.gov (United States)

    Bettiga, Arianna; Aureli, Massimo; Colciago, Giorgia; Murdica, Valentina; Moschini, Marco; Lucianò, Roberta; Canals, Daniel; Hannun, Yusuf; Hedlund, Petter; Lavorgna, Giovanni; Colombo, Renzo; Bassi, Rosaria; Samarani, Maura; Montorsi, Francesco; Salonia, Andrea; Benigni, Fabio

    2017-01-01

    The inhibitory effects demonstrated by activation of cannabinoid receptors (CB) on cancer proliferation and migration may also play critical roles in controlling bladder cancer (BC). CB expression on human normal and BC specimens was tested by immunohistochemistry. Human BC cells RT4 and RT112 were challenged with CB agonists and assessed for proliferation, apoptosis, and motility. Cellular sphingolipids (SL) constitution and metabolism were evaluated after metabolic labelling. CB1-2 were detected in BC specimens, but only CB2 was more expressed in the tumour. Both cell lines expressed similar CB2. Exposure to CB2 agonists inhibited BC growth, down-modulated Akt, induced caspase 3-activation and modified SL metabolism. Baseline SL analysis in cell lines showed differences linked to unique migratory behaviours and cytoskeletal re-arrangements. CB2 activation changed the SL composition of more aggressive RT112 cells by reducing (p < 0.01) Gb3 ganglioside (−50 ± 3%) and sphingosine 1-phosphate (S1P, −40 ± 4%), which ended up to reduction in cell motility (−46 ± 5%) with inhibition of p-SRC. CB2-selective antagonists, gene silencing and an inhibitor of SL biosynthesis partially prevented CB2 agonist-induced effects on cell viability and motility. CB2 activation led to ceramide-mediated BC cell apoptosis independently of SL constitutive composition, which instead was modulated by CB2 agonists to reduce cell motility. PMID:28191815

  3. Interrogating Androgen Receptor Mediated Gene Expression and Tumor Progression by Molecular Imaging

    Science.gov (United States)

    2005-10-01

    Edwards J, Bartlett JM . The androgen receptor and signal-transduc- tion pathways in hormone-refractory prostate cancer. Part 2. Androgen- One of the key...34. Kim J, Jia L, Tilley WD, Coetzee GA. Dynamic methylation of histone modulation of androgen receptor transcriptional activity by the nuclear H3 at

  4. Natural Modulators of Endosomal Toll-Like Receptor-Mediated Psoriatic Skin Inflammation

    Directory of Open Access Journals (Sweden)

    Chao-Yang Lai

    2017-01-01

    Full Text Available Psoriasis is a chronic inflammatory autoimmune disease that can be initiated by excessive activation of endosomal toll-like receptors (TLRs, particularly TLR7, TLR8, and TLR9. Therefore, inhibitors of endosomal TLR activation are being investigated for their ability to treat this disease. The currently approved biological drugs adalimumab, etanercept, infliximab, ustekinumab, ixekizumab, and secukizumab are antibodies against effector cytokines that participate in the initiation and development of psoriasis. Several immune modulatory oligonucleotides and small molecular weight compounds, including IMO-3100, IMO-8400, and CPG-52364, that block the interaction between endosomal TLRs and their ligands are under clinical investigation for their effectiveness in the treatment of psoriasis. In addition, several chemical compounds, including AS-2444697, PF-05387252, PF-05388169, PF-06650833, ML120B, and PHA-408, can inhibit TLR signaling. Although these compounds have demonstrated anti-inflammatory activity in animal models, their therapeutic potential for the treatment of psoriasis has not yet been tested. Recent studies demonstrated that natural compounds derived from plants, fungi, and bacteria, including mustard seed, Antrodia cinnamomea extract, curcumin, resveratrol, thiostrepton, azithromycin, and andrographolide, inhibited psoriasis-like inflammation induced by the TLR7 agonist imiquimod in animal models. These natural modulators employ different mechanisms to inhibit endosomal TLR activation and are administered via different routes. Therefore, they represent candidate psoriasis drugs and might lead to the development of new treatment options.

  5. Serotonin(2) receptors mediate respiratory recovery after cervical spinal cord hemisection in adult rats.

    Science.gov (United States)

    Zhou, S Y; Basura, G J; Goshgarian, H G

    2001-12-01

    The aim of the present study was to specifically investigate the involvement of serotonin [5-hydroxytryptamine (5-HT(2))] receptors in 5-HT-mediated respiratory recovery after cervical hemisection. Experiments were conducted on C(2) spinal cord-hemisected, anesthetized (chloral hydrate, 400 mg/kg ip), vagotomized, pancuronium- paralyzed, and artificially ventilated female Sprague-Dawley rats in which CO(2) levels were monitored and maintained. Twenty-four hours after spinal hemisection, the ipsilateral phrenic nerve displayed no respiratory-related activity indicative of a functionally complete hemisection. Intravenous administration of the 5-HT(2A/2C)-receptor agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) induced respiratory-related activity in the phrenic nerve ipsilateral to hemisection under conditions in which CO(2) was maintained at constant levels and augmented the activity induced under conditions of hypercapnia. The effects of DOI were found to be dose dependent, and the recovery of activity could be maintained for up to 2 h after a single injection. DOI-induced recovery was attenuated by the 5-HT(2)-receptor antagonist ketanserin but not with the 5-HT(2C)-receptor antagonist RS-102221, suggesting that 5-HT(2A) and not necessarily 5-HT(2C) receptors may be involved in the induction of respiratory recovery after cervical spinal cord injury.

  6. Nortriptyline induces mitochondria and death receptor-mediated apoptosis in bladder cancer cells and inhibits bladder tumor growth in vivo.

    Science.gov (United States)

    Yuan, Sheau-Yun; Cheng, Chen-Li; Ho, Hao-Chung; Wang, Shian-Shiang; Chiu, Kun-Yuan; Su, Chung-Kuang; Ou, Yen-Chuan; Lin, Chi-Chen

    2015-08-15

    Nortriptyline (NTP), an antidepressant, has antitumor effects on some human cancer cells, but its effect on human bladder cancer cells is not known. In this study, we used a cell viability assay to demonstrate that NTP is cytotoxic to human TCCSUP and mouse MBT-2 bladder cancer cells in a concentration and time-dependent manner. We also performed cell cycle analysis, annexin V and mitochondrial membrane potential assays, and Western blot analysis to show that NTP inhibits cell growth in these cells by inducing both mitochondria-mediated and death receptor-mediated apoptosis. Specifically, NTP increases the expression of Fas, FasL, FADD, Bax, Bak, and cleaved forms of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase. In addition, NTP decreases the expression of Bcl-2, Bcl-xL, BH3 interacting domain death agonist, X-linked inhibitor of apoptosis protein, and survivin. Furthermore, NTP-induced apoptosis is associated with reactive oxygen species (ROS) production, which can be reduced by antioxidants, such as N-acetyl-L-cysteine. Finally, we showed that NTP suppresses tumor growth in mice inoculated with MBT-2 cells. Collectively, our results suggest that NTP induces both intrinsic and extrinsic apoptosis in human and mouse bladder cancer cells and that it may be a clinically useful chemotherapeutic agent for bladder cancer in humans. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. An special epithelial staining agents: folic acid receptor-mediated diagnosis (FRD) effectively and conveniently screen patients with cervical cancer.

    Science.gov (United States)

    Lu, Meng-Han; Hu, Ling-Yun; Du, Xin-Xin; Yang, Min; Zhang, Wei-Yi; Huang, Ke; Li, Li-An; Jiang, Shu-Fang; Li, Ya-Li

    2015-01-01

    High-quality screening with cytology has markedly reduced mortality from cervical cancer. However, it needs experienced pathologists to review and make the final decisions. We have developed folic acid receptor-mediated diagnosis (FRD) kits to effectively and conveniently screen patients with cervical cancer. We conduct present study aim to assess clinical significances of FRD in screening cervical cancer. A total of 169 patients were enrolled at Chinese People's liberation Army (PLA) general hospital. We compared diagnostic significances of FRD with thinprep cytology test (TCT). Meanwhile, colposcopy was also performed to confirm any lesion suspicious for cervical cancer. The sensitivity and specificity of FRD were 71.93% and 66.07% in diagnosis cervical cancer, respectively. Meanwhile, the positive predictive values (PPV), negative predictive values (NPV), Youden index were 51.90%, 82.22%, 0.38, respectively. On the other hand, the sensitivity and specificity of TCT in diagnosis cervical cancer were 73.68% and 61.61% respectively. PPV, NPV and Youden index for TCT were 49.41%, 82.14% and 0.35 respectively. Overall, FRD have high values of sensitivity, specificity and Youden index. However, this difference failed to statistical significance. FRD have comparable diagnostic significance with TCT. Therefore, FRD might serve as one effective method to screen cervical cancer. Especially for those patients living in remote regions of China, where cytology was unavailable.

  8. The Influence of Receptor-Mediated Interactions on Reaction-Diffusion Mechanisms of Cellular Self-organisation

    KAUST Repository

    Klika, Václav

    2011-11-10

    Understanding the mechanisms governing and regulating self-organisation in the developing embryo is a key challenge that has puzzled and fascinated scientists for decades. Since its conception in 1952 the Turing model has been a paradigm for pattern formation, motivating numerous theoretical and experimental studies, though its verification at the molecular level in biological systems has remained elusive. In this work, we consider the influence of receptor-mediated dynamics within the framework of Turing models, showing how non-diffusing species impact the conditions for the emergence of self-organisation. We illustrate our results within the framework of hair follicle pre-patterning, showing how receptor interaction structures can be constrained by the requirement for patterning, without the need for detailed knowledge of the network dynamics. Finally, in the light of our results, we discuss the ability of such systems to pattern outside the classical limits of the Turing model, and the inherent dangers involved in model reduction. © 2011 Society for Mathematical Biology.

  9. Cryptococcus neoformans Is Internalized by Receptor-Mediated or ‘Triggered’ Phagocytosis, Dependent on Actin Recruitment

    Science.gov (United States)

    Guerra, Caroline Rezende; Seabra, Sergio Henrique; de Souza, Wanderley; Rozental, Sonia

    2014-01-01

    Cryptococcosis by the encapsulated yeast Cryptococcus neoformans affects mostly immunocompromised individuals and is a frequent neurological complication in AIDS patients. Recent studies support the idea that intracellular survival of Cryptococcus yeast cells is important for the pathogenesis of cryptococcosis. However, the initial steps of Cryptococcus internalization by host cells remain poorly understood. Here, we investigate the mechanism of Cryptococcus neoformans phagocytosis by peritoneal macrophages using confocal and electron microscopy techniques, as well as flow cytometry quantification, evaluating the importance of fungal capsule production and of host cell cytoskeletal elements for fungal phagocytosis. Electron microscopy analyses revealed that capsular and acapsular strains of C. neoformans are internalized by macrophages via both ‘zipper’ (receptor-mediated) and ‘trigger’ (membrane ruffle-dependent) phagocytosis mechanisms. Actin filaments surrounded phagosomes of capsular and acapsular yeasts, and the actin depolymerizing drugs cytochalasin D and latrunculin B inhibited yeast internalization and actin recruitment to the phagosome area. In contrast, nocodazole and paclitaxel, inhibitors of microtubule dynamics decreased internalization but did not prevent actin recruitment to the site of phagocytosis. Our results show that different uptake mechanisms, dependent on both actin and tubulin dynamics occur during yeast internalization by macrophages, and that capsule production does not affect the mode of Cryptococcus uptake by host cells. PMID:24586631

  10. Regulation of VH replacement by B cell receptor-mediated signaling in human immature B cells.

    Science.gov (United States)

    Liu, Jing; Lange, Miles D; Hong, Sang Yong; Xie, Wanqin; Xu, Kerui; Huang, Lin; Yu, Yangsheng; Ehrhardt, Götz R A; Zemlin, Michael; Burrows, Peter D; Su, Kaihong; Carter, Robert H; Zhang, Zhixin

    2013-06-01

    VH replacement provides a unique RAG-mediated recombination mechanism to edit nonfunctional IgH genes or IgH genes encoding self-reactive BCRs and contributes to the diversification of Ab repertoire in the mouse and human. Currently, it is not clear how VH replacement is regulated during early B lineage cell development. In this article, we show that cross-linking BCRs induces VH replacement in human EU12 μHC(+) cells and in the newly emigrated immature B cells purified from peripheral blood of healthy donors or tonsillar samples. BCR signaling-induced VH replacement is dependent on the activation of Syk and Src kinases but is inhibited by CD19 costimulation, presumably through activation of the PI3K pathway. These results show that VH replacement is regulated by BCR-mediated signaling in human immature B cells, which can be modulated by physiological and pharmacological treatments.

  11. Mechanisms of G Protein-Coupled Estrogen Receptor-Mediated Spinal Nociception

    DEFF Research Database (Denmark)

    Deliu, Elena; Brailoiu, G. Cristina; Arterburn, Jeffrey B.

    2012-01-01

    in spinal nociceptive processing. Intrathecal challenging of mice with the GPER agonist G-1 results in pain-related behaviors. GPER antagonism with G15 reduces the G-1-induced response. Electrophysiological recordings from superficial dorsal horn neurons indicate neuronal membrane depolarization with G-1......Human and animal studies suggest that estrogens are involved in the processing of nociceptive sensory information and analgesic responses in the central nervous system. Rapid pronociceptive estrogenic effects have been reported, some of which likely involve G protein-coupled estrogen receptor (GPER......) activation. Membrane depolarization and increases in cytosolic calcium and reactive oxygen species (ROS) levels are markers of neuronal activation, underlying pain sensitization in the spinal cord. Using behavioral, electrophysiological, and fluorescent imaging studies, we evaluated GPER involvement...

  12. Cellular mechanisms of the 5-HT7 receptor-mediated signaling

    OpenAIRE

    Guseva, Daria; Wirth, Alexander; Ponimaskin, Evgeni

    2014-01-01

    Serotonin (5-hydroxytryptamine or 5-HT) is an important neurotransmitter regulating a wide range of physiological and pathological functions via activation of heterogeneously expressed 5-HT receptors. The 5-HT7 receptor is one of the most recently described members of the 5-HT receptor family. Functionally, 5-HT7 receptor is associated with a number of physiological and pathological responses, including serotonin-induced phase shifting of the circadian rhythm, control of memory as well as loc...

  13. Nicotinic receptor mediates nitric oxide synthase expression in the rat gastric myenteric plexus.

    OpenAIRE

    1998-01-01

    The mechanism that regulates the synthesis of nitric oxide synthase (NOS), a key enzyme responsible for NO production in the myenteric plexus, remains unknown. We investigated the roles of the vagal nerve and nicotinic synapses in the mediation of NOS synthesis in the gastric myenteric plexus in rats. Truncal vagotomy and administration of hexamethonium significantly reduced nonadrenergic, noncholinergic relaxation, the catalytic activity of NOS, the number of NOS-immunoreactive cells, and th...

  14. Metabotropic Glutamate Receptor-Mediated Long-Term Depression: Molecular Mechanisms

    OpenAIRE

    Gladding, Clare M.; Fitzjohn, Stephen M; Molnár, Elek

    2009-01-01

    The ability to modify synaptic transmission between neurons is a fundamental process of the nervous system that is involved in development, learning, and disease. Thus, synaptic plasticity is the ability to bidirectionally modify transmission, where long-term potentiation and long-term depression (LTD) represent the best characterized forms of plasticity. In the hippocampus, two main forms of LTD coexist that are mediated by activation of either N-methyl-d-aspartic acid receptors (NMDARs) or ...

  15. Nicotinic acetylcholine receptor-mediated calcium signaling in the nervous system

    Institute of Scientific and Technical Information of China (English)

    Jian-xin SHEN; Jerrel L YAKEL

    2009-01-01

    Based on the composition of the five subunits forming functional neuronal nicotinic acetylcholine receptors (nAChRs), they are grouped into either heteromeric (comprising both α and β subunits) or homomeric (comprising only α subunits) recep-tors. The nAChRs are known to be differentially permeable to calcium ions, with the α7 nAChR subtype having one of the highest permeabilities to calcium. Calcium influx through nAChRs, particularly through the α-bungarotoxin-sensitive α7-containing nAChRs, is a very efficient way to raise cytoplasmic calcium levels. The activation of nAChRs can mediate three types of cytoplasmic calcium signals: (1) direct calcium influx through the nAChRs, (2) indirect calcium influx through voltage-dependent calcium channels (VDCCs) which are activated by the nAChR-mediated depolarization, and (3) calcium-induced calcium release (CICR) (triggered by the first two sources) from the endoplasmic reticulum (ER) through the ryanodine receptors and inositol (1,4,5)-triphosphate receptors (IP3Rs). Downstream signaling events mediated by nAChR-mediated calcium responses can be grouped into instantaneous effects (such as neurotransmitter release, which can occur in milliseconds after nAChR activation), short-term effects (such as the recovery of nAChR desensitization through cellular signaling cascades), and long-term effects (such as neuroprotection via gene expression). In addition, nAChR activity can be regulated by cytoplasmic calcium levels, suggesting a complex reciprocal relationship. Further advances in imaging techniques, animal models, and more potent and subtype-selective ligands for neuronal nAChRs would help in understand-ing the neuronal nAChR-mediated calcium signaling, and lead to the development of improved therapeutic treatments.

  16. A model to estimate the oestrogen receptor mediated effects from exposure to soy isoflavones in food.

    Science.gov (United States)

    Safford, Bob; Dickens, Andrea; Halleron, Nadine; Briggs, David; Carthew, Philip; Baker, Valerie

    2003-10-01

    The advantages that regular consumption of a diet containing soy may have on human health have been enshrined in a major health claim that has been approved by the Food and Drug Administration in the USA, regarding potential protection from heart disease by soy. This could have a major influence on the dietary consumption patterns of soy for consumers and lead to the development of soy enriched foods to enable consumers to achieve the benefits thought to be associated with increased soy consumption in a Western diet. If an increase in soy consumption is beneficial to particular disease conditions, there is always the possibility that there will be effects other than those that are desirable. For soy-containing foods there has been concern that the phytoestrogen content of soy, which is composed of several isoflavones, could be a separate health issue, due to the oestrogen-like activity of isoflavones. To address this, a method has been developed to estimate, relative to 17-beta oestradiol, the activity of the common isoflavones present in soy phytoestrogens, based on their binding to and transcriptional activation of the major oestrogen receptor sub-types alpha and beta. Using this approach, the additional oestrogen-like activity that would be expected from inclusion of soy supplemented foodstuffs in a Western diet, can be determined for different sub-populations, who may have different susceptibilities to the potential for the unwanted biological effects occurring with consumption of soy enriched foods. Because of the theoretical nature of this model, and the controversy over the nature of whether some of the oestrogen-like effects of phytoestrogens are adverse, the biological effects of soy isoflavones and their potential for adverse effects in man, is also reviewed. The question that is critical to the long term safe use of foods enriched in soy is, which observed biological effects in animal studies are likely to also occur in man and whether these would have

  17. A possibly sigma-1 receptor mediated role of dimethyltryptamine in tissue protection, regeneration, and immunity.

    Science.gov (United States)

    Frecska, Ede; Szabo, Attila; Winkelman, Michael J; Luna, Luis E; McKenna, Dennis J

    2013-09-01

    N,N-dimethyltryptamine (DMT) is classified as a naturally occurring serotonergic hallucinogen of plant origin. It has also been found in animal tissues and regarded as an endogenous trace amine transmitter. The vast majority of research on DMT has targeted its psychotropic/psychedelic properties with less focus on its effects beyond the nervous system. The recent discovery that DMT is an endogenous ligand of the sigma-1 receptor may shed light on yet undiscovered physiological mechanisms of DMT activity and reveal some of its putative biological functions. A three-step active uptake process of DMT from peripheral sources to neurons underscores a presumed physiological significance of this endogenous hallucinogen. In this paper, we overview the literature on the effects of sigma-1 receptor ligands on cellular bioenergetics, the role of serotonin, and serotoninergic analogues in immunoregulation and the data regarding gene expression of the DMT synthesizing enzyme indolethylamine-N-methyltransferase in carcinogenesis. We conclude that the function of DMT may extend central nervous activity and involve a more universal role in cellular protective mechanisms. Suggestions are offered for future directions of indole alkaloid research in the general medical field. We provide converging evidence that while DMT is a substance which produces powerful psychedelic experiences, it is better understood not as a hallucinogenic drug of abuse, but rather an agent of significant adaptive mechanisms that can also serve as a promising tool in the development of future medical therapies.

  18. Pax6 represses androgen receptor-mediated transactivation by inhibiting recruitment of the coactivator SPBP.

    Directory of Open Access Journals (Sweden)

    Julianne Elvenes

    Full Text Available The androgen receptor (AR has a central role in development and maintenance of the male reproductive system and in the etiology of prostate cancer. The transcription factor Pax6 has recently been reported to act as a repressor of AR and to be hypermethylated in prostate cancer cells. SPBP is a transcriptional regulator that previously has been shown to enhance the activity of Pax6. In this study we have identified SPBP to act as a transcriptional coactivator of AR. We also show that Pax6 inhibits SPBP-mediated enhancement of AR activity on the AR target gene probasin promoter, a repression that was partly reversed by increased expression of SPBP. Enhanced expression of Pax6 reduced the amount of SPBP associated with the probasin promoter when assayed by ChIP in HeLa cells. We mapped the interaction between both AR and SPBP, and AR and Pax6 to the DNA-binding domains of the involved proteins. Further binding studies revealed that Pax6 and SPBP compete for binding to AR. These results suggest that Pax6 represses AR activity by displacing and/or inhibiting recruitment of coactivators to AR target promoters. Understanding the mechanism for inhibition of AR coactivators can give rise to molecular targeted drugs for treatment of prostate cancer.

  19. Genetic disruption of oncogenic Kras sensitizes lung cancer cells to Fas receptor-mediated apoptosis.

    Science.gov (United States)

    Mou, Haiwei; Moore, Jill; Malonia, Sunil K; Li, Yingxiang; Ozata, Deniz M; Hough, Soren; Song, Chun-Qing; Smith, Jordan L; Fischer, Andrew; Weng, Zhiping; Green, Michael R; Xue, Wen

    2017-04-04

    Genetic lesions that activate KRAS account for ∼30% of the 1.6 million annual cases of lung cancer. Despite clinical need, KRAS is still undruggable using traditional small-molecule drugs/inhibitors. When oncogenic Kras is suppressed by RNA interference, tumors initially regress but eventually recur and proliferate despite suppression of Kras Here, we show that tumor cells can survive knockout of oncogenic Kras, indicating the existence of Kras-independent survival pathways. Thus, even if clinical KRAS inhibitors were available, resistance would remain an obstacle to treatment. Kras-independent cancer cells exhibit decreased colony formation in vitro but retain the ability to form tumors in mice. Comparing the transcriptomes of oncogenic Kras cells and Kras knockout cells, we identified 603 genes that were specifically up-regulated in Kras knockout cells, including the Fas gene, which encodes a cell surface death receptor involved in physiological regulation of apoptosis. Antibodies recognizing Fas receptor efficiently induced apoptosis of Kras knockout cells but not oncogenic Kras-expressing cells. Increased Fas expression in Kras knockout cells was attributed to decreased association of repressive epigenetic marks at the Fas promoter. Concordant with this observation, treating oncogenic Kras cells with histone deacetylase inhibitor and Fas-activating antibody efficiently induced apoptosis, thus bypassing the need to inhibit Kras. Our results suggest that activation of Fas could be exploited as an Achilles' heel in tumors initiated by oncogenic Kras.

  20. Bidirectional regulation of angiogenesis by phytoestrogens through estrogen receptor-mediated signaling networks.

    Science.gov (United States)

    Liu, Hai-Xin; Wang, Yu; Lu, Qing; Yang, Ming-Zhu; Fan, Guan-Wei; Karas, Richard H; Gao, Xiu-Mei; Zhu, Yan

    2016-04-01

    Sex hormone estrogen is one of the most active intrinsic angiogenesis regulators; its therapeutic use has been limited due to its carcinogenic potential. Plant-derived phytoestrogens are attractive alternatives, but reports on their angiogenic activities often lack in-depth analysis and sometimes are controversial. Herein, we report a data-mining study with the existing literature, using IPA system to classify and characterize phytoestrogens based on their angiogenic properties and pharmacological consequences. We found that pro-angiogenic phytoestrogens functioned predominantly as cardiovascular protectors whereas anti-angiogenic phytoestrogens played a role in cancer prevention and therapy. This bidirectional regulation were shown to be target-selective and, for the most part, estrogen-receptor-dependent. The transactivation properties of ERα and ERβ by phytoestrogens were examined in the context of angiogenesis-related gene transcription. ERα and ERβ were shown to signal in opposite ways when complexed with the phytoestrogen for bidirectional regulation of angiogenesis. With ERα, phytoestrogen activated or inhibited transcription of some angiogenesis-related genes, resulting in the promotion of angiogenesis, whereas, with ERβ, phytoestrogen regulated transcription of angiogenesis-related genes, resulting in inhibition of angiogenesis. Therefore, the selectivity of phytoestrogen to ERα and ERβ may be critical in the balance of pro- or anti-angiogenesis process.

  1. Steroid and xenobiotic receptor-mediated effects of bisphenol A on human osteoblasts.

    Science.gov (United States)

    Miki, Yasuhiro; Hata, Shuko; Nagasaki, Shuji; Suzuki, Takashi; Ito, Kiyoshi; Kumamoto, Hiroyuki; Sasano, Hironobu

    2016-06-15

    Bisphenol A, one of the industrial chemicals used in plastics and in the coating of dishes and medical equipment, behaves as an endocrine disruptor in the human body. Bisphenol A can bind directly to several types of nuclear receptors, including steroid and xenobiotic receptor (SXR). SXR plays an important role in bone metabolism through the activation of osteoblasts in vitro, but SXR protein localization has not been reported in bone tissues. Additionally, it is not known whether bisphenol A acts on osteoblasts through SXR activation. Therefore, in this study, we first examined the immunolocalization of the SXR protein in human adult and fetal bone tissues. We then examined the effects of bisphenol A on human osteoblasts in vitro. SXR immunoreactivity was detected in osteoblasts, but not in osteoclasts, of both adult and fetal bone tissues. In fetal bone tissues, the mesenchymal cells or fetal connective tissue were also positive for SXR immunoreactivity. Expression of SXR target genes (tsukushi, matrilin-2, and CYP3A4) and SXR response element-luciferase activity were increased by bisphenol A treatment in normal osteoblasts transfected with SXR (hFOB/SXR) and in osteoblast-like cells (MG-63). Bisphenol A also stimulated cell proliferation and collagen accumulation in hFOB/SXR cells. These results suggest that, as in other tissues, SXR plays important roles in bone metabolism and fetal bone development and that bisphenol A may disturb bone homeostasis in both adult and fetus through SXR.

  2. Inhibitory nature of tiagabine-augmented GABAA receptor-mediated depolarizing responses in hippocampal pyramidal cells.

    Science.gov (United States)

    Jackson, M F; Esplin, B; Capek, R

    1999-03-01

    Tiagabine is a potent GABA uptake inhibitor with demonstrated anticonvulsant activity. GABA uptake inhibitors are believed to produce their anticonvulsant effects by prolonging the postsynaptic actions of GABA, released during episodes of neuronal hyperexcitability. However, tiagabine has recently been reported to facilitate the depolarizing actions of GABA in the CNS of adult rats following the stimulation of inhibitory pathways at a frequency (100 Hz) intended to mimic interneuronal activation during epileptiform activity. In the present study, we performed extracellular and whole cell recordings from CA1 pyramidal neurons in rat hippocampal slices to examine the functional consequences of tiagabine-augmented GABA-mediated depolarizing responses. Orthodromic population spikes (PSs), elicited from the stratum radiatum, were inhibited following the activation of recurrent inhibitory pathways by antidromic conditioning stimulation of the alveus, which consisted of either a single stimulus or a train of stimuli delivered at high-frequency (100 Hz, 200 ms). The inhibition of orthodromic PSs produced by high-frequency conditioning stimulation (HFS), which was always of much greater strength and duration than that produced by a single conditioning stimulus, was greatly enhanced following the bath application of tiagabine (2-100 microM). Thus, in the presence of tiagabine (20 microM), orthodromic PSs, evoked 200 and 800 ms following HFS, were inhibited to 7.8 +/- 2.6% (mean +/- SE) and 34.4 +/- 18.5% of their unconditioned amplitudes compared with only 35.4 +/- 12.7% and 98.8 +/- 12.4% in control. Whole cell recordings revealed that the bath application of tiagabine (20 microM) either caused the appearance or greatly enhanced the amplitude of GABA-mediated depolarizing responses (DR). Excitatory postsynaptic potentials (EPSPs) evoked from stratum radiatum at time points that coincided with the DR were inhibited to below the threshold for action-potential firing

  3. Differential GABAB-receptor-mediated effects in perisomatic- and dendrite-targeting parvalbumin interneurons.

    Science.gov (United States)

    Booker, Sam A; Gross, Anna; Althof, Daniel; Shigemoto, Ryuichi; Bettler, Bernhard; Frotscher, Michael; Hearing, Matthew; Wickman, Kevin; Watanabe, Masahiko; Kulik, Ákos; Vida, Imre

    2013-05-01

    Inhibitory parvalbumin-containing interneurons (PVIs) control neuronal discharge and support the generation of theta- and gamma-frequency oscillations in cortical networks. Fast GABAergic input onto PVIs is crucial for their synchronization and oscillatory entrainment, but the role of metabotropic GABA(B) receptors (GABA(B)Rs) in mediating slow presynaptic and postsynaptic inhibition remains unknown. In this study, we have combined high-resolution immunoelectron microscopy, whole-cell patch-clamp recording, and computational modeling to investigate the subcellular distribution and effects of GABA(B)Rs and their postsynaptic effector Kir3 channels in rat hippocampal PVIs. Pre-embedding immunogold labeling revealed that the receptors and channels localize at high levels to the extrasynaptic membrane of parvalbumin-immunoreactive dendrites. Immunoreactivity for GABA(B)Rs was also present at lower levels on PVI axon terminals. Whole-cell recordings further showed that synaptically released GABA in response to extracellular stimulation evokes large GABA(B)R-mediated slow IPSCs in perisomatic-targeting (PT) PVIs, but only small or no currents in dendrite-targeting (DT) PVIs. In contrast, paired recordings demonstrated that GABA(B)R activation results in presynaptic inhibition at the output synapses of both PT and DT PVIs, but more strongly in the latter. Finally, computational analysis indicated that GABA(B) IPSCs can phasically modulate the discharge of PT interneurons at theta frequencies. In summary, our results show that GABA(B)Rs differentially mediate slow presynaptic and postsynaptic inhibition in PVIs and can contribute to the dynamic modulation of their activity during oscillations. Furthermore, these data provide evidence for a compartment-specific molecular divergence of hippocampal PVI subtypes, suggesting that activation of GABA(B)Rs may shift the balance between perisomatic and dendritic inhibition.

  4. Muscarinic receptor binding and muscarinic receptor-mediated inhibition of adenylate cyclase in rat brain myelin

    Energy Technology Data Exchange (ETDEWEB)

    Larocca, J.N.; Ledeen, R.W.; Dvorkin, B.; Makman, M.H.

    1987-12-01

    High-affinity muscarinic cholinergic receptors were detected in myelin purified from rat brain stem with use of the radioligands /sup 3/H-N-methylscopolamine (/sup 3/H-NMS), /sup 3/H-quinuclidinyl benzilate (/sup 3/H-QNB), and /sup 3/H-pirenzepine. /sup 3/H-NMS binding was also present in myelin isolated from corpus callosum. In contrast, several other receptor types, including alpha 1- and alpha 2-adrenergic receptors, present in the starting brain stem, were not detected in myelin. Based on Bmax values from Scatchard analyses, /sup 3/H-pirenzepine, a putative M1 selective ligand, bound to about 25% of the sites in myelin labeled by /sup 3/H-NMS, a nonselective ligand that binds to both M1 and M2 receptor subtypes. Agonist affinity for /sup 3/H-NMS binding sites in myelin was markedly decreased by Gpp(NH)p, indicating that a major portion of these receptors may be linked to a second messenger system via a guanine-nucleotide regulatory protein. Purified myelin also contained adenylate cyclase activity; this activity was stimulated several fold by forskolin and to small but significant extents by prostaglandin E1 and the beta-adrenergic agonist isoproterenol. Myelin adenylate cyclase activity was inhibited by carbachol and other muscarinic agonists; this inhibition was blocked by the antagonist atropine. Levels in myelin of muscarinic receptors were 20-25% and those of forskolin-stimulated adenylate cyclase 10% of the values for total particulate fraction of whole brain stem. These levels in myelin are appreciably greater than would be predicted on the basis of contamination. Also, additional receptors and adenylate cyclase, added by mixing nonmyelin tissue with whole brain stem, were quantitatively removed during the purification procedure.

  5. NMDA receptors mediate stimulus-timing-dependent plasticity and neural synchrony in the dorsal cochlear nucleus

    Directory of Open Access Journals (Sweden)

    Roxana A Stefanescu

    2015-11-01

    Full Text Available Auditory information relayed by auditory nerve fibers and somatosensory information relayed by granule cell parallel fibers converge on the fusiform cells (FCs of the dorsal cochlear nucleus, the first brain station of the auditory pathway. In vitro, parallel fiber synapses on FCs exhibit spike-timing-dependent plasticity with Hebbian learning rules, partially mediated by the NMDA receptor (NMDAr. Well-timed bimodal auditory-somatosensory stimulation, in vivo equivalent of spike-timing-dependent plasticity, can induce stimulus-timing-dependent plasticity (StTDP of the FCs spontaneous and tone-evoked firing rates. In healthy guinea pigs, the resulting distribution of StTDP learning rules across a FC neural population is dominated by a Hebbian profile while anti-Hebbian, suppressive and enhancing LRs are less frequent. In this study, we investigate in vivo, the NMDAr contribution to FC baseline activity and long term plasticity. We find that blocking the NMDAr decreases the synchronization of FC- spontaneous activity and mediates differential modulation of FC rate-level functions such that low, and high threshold units are more likely to increase, and decrease, respectively, their maximum amplitudes. Three significant alterations in mean learning-rule profiles were identified: transitions from an initial Hebbian profile towards (1 an anti-Hebbian and (2 a suppressive profile, and (3 transitions from an anti-Hebbian to a Hebbian profile. FC units preserving their learning rules showed instead, NMDAr-dependent plasticity to unimodal acoustic stimulation, with persistent depression of tone-evoked responses changing to persistent enhancement following the NMDAr antagonist. These results reveal a crucial role of the NMDAr in mediating FC baseline activity and long-term plasticity which have important implications for signal processing and auditory pathologies related to maladaptive plasticity of dorsal cochlear nucleus circuitry.

  6. Interaction of medullary P2 and glutamate receptors mediates the vasodilation in the hindlimb of rat.

    Science.gov (United States)

    Korim, Willian Seiji; Ferreira-Neto, Marcos L; Pedrino, Gustavo R; Pilowsky, Paul M; Cravo, Sergio L

    2012-12-01

    In the nucleus tractus solitarii (NTS) of rats, blockade of extracellular ATP breakdown to adenosine reduces arterial blood pressure (AP) increases that follow stimulation of the hypothalamic defense area (HDA). The effects of ATP on NTS P2 receptors, during stimulation of the HDA, are still unclear. The aim of this study was to determine whether activation of P2 receptors in the NTS mediates cardiovascular responses to HDA stimulation. Further investigation was taken to establish if changes in hindlimb vascular conductance (HVC) elicited by electrical stimulation of the HDA, or activation of P2 receptors in the NTS, are relayed in the rostral ventrolateral medulla (RVLM); and if those responses depend on glutamate release by ATP acting on presynaptic terminals. In anesthetized and paralyzed rats, electrical stimulation of the HDA increased AP and HVC. Blockade of P2 or glutamate receptors in the NTS, with bilateral microinjections of suramin (10 mM) or kynurenate (50 mM) reduced only the evoked increase in HVC by 75 % or more. Similar results were obtained with the blockade combining both antagonists. Blockade of P2 and glutamate receptors in the RVLM also reduced the increases in HVC to stimulation of the HDA by up to 75 %. Bilateral microinjections of kynurenate in the RVLM abolished changes in AP and HVC to injections of the P2 receptor agonist α,β-methylene ATP (20 mM) into the NTS. The findings suggest that HDA-NTS-RVLM pathways in control of HVC are mediated by activation of P2 and glutamate receptors in the brainstem in alerting-defense reactions.

  7. Adiponectin increases secretion of rat submandibular gland via adiponectin receptors-mediated AMPK signaling.

    Directory of Open Access Journals (Sweden)

    Chong Ding

    Full Text Available Adiponectin and adiponectin receptors (AdipoR1/2 are expressed in various tissues and are involved in the regulation of multiple functions such as energy metabolism and inflammatory responses. However, the effect of adiponectin and AdipoRs in submandibular glands has not been fully evaluated. In the present study, we found that mRNA and protein of both adiponectin and AdipoR1/2 were expressed in rat submandibular glands and in the SMG-C6 cell line, as evidenced by RT-PCR and Western blot analysis. Immunofluorescence staining showed that adiponectin was diffused in the cytoplasm, while AdipoR1/2 was concentrated in the membrane of acinar cells. Saliva flow was significantly increased by full length adiponectin (fAd or globular adiponectin (gAd perfusion in isolated rat submandibular glands. 5-Aminoimidazole-4-carboxamide-1-4-ribofuranoside (AICAR, an adenosine monophosphate activated protein kinase (AMPK activator, also increased saliva secretion. fAd, gAd, and AICAR all increased the average width of apical tight junctions in perfused submandibular glands, and decreased transepithelial electrical resistance (TER in SMG-C6 cells, suggesting that adiponectin promoted secretion by modulating paracellular permeability. fAd and gAd increased p-AMPK levels, while AraA, an AMPK antagonist, abolished fAd- and gAd-induced changes in secretion, tight junction ultrastructure, and TER. Moreover, both AdipoR1 and AdipoR2 were required for fAd- or gAd-induced p-AMPK and TER responses, suggesting from their inhibition following AdipoR1 or AdipoR2 knockdown, and co-knockdown of AdipoRs by RNA interference. Our results suggest that adiponectin functions as a promoter of salivary secretion in rat submandibular glands via activation of AdipoRs, AMPK, and paracellular permeability.

  8. Basolateral amygdala CB1 cannabinoid receptors mediate nicotine-induced place preference.

    Science.gov (United States)

    Hashemizadeh, Shiva; Sardari, Maryam; Rezayof, Ameneh

    2014-06-03

    In the present study, the effects of bilateral microinjections of cannabinoid CB1 receptor agonist and antagonist into the basolateral amygdala (intra-BLA) on nicotine-induced place preference were examined in rats. A conditioned place preference (CPP) apparatus was used for the assessment of rewarding effects of the drugs in adult male Wistar rats. Subcutaneous (s.c.) administration of nicotine (0.2mg/kg) induced a significant CPP, without any effect on the locomotor activity during the testing phase. Intra-BLA microinjection of a non-selective cannabinoid CB1/CB2 receptor agonist, WIN 55,212-2 (0.1-0.5 μg/rat) with an ineffective dose of nicotine (0.1mg/kg, s.c.) induced a significant place preference. On the other hand, intra-BLA administration of AM251 (20-60 ng/rat), a selective cannabinoid CB1 receptor antagonist inhibited the acquisition of nicotine-induced place preference. It should be considered that the microinjection of the same doses of WIN 55,212-2 or AM251 into the BLA, by itself had no effect on the CPP score. The administration of a higher dose of AM251 (60 ng/rat) during the acquisition decreased the locomotor activity of animals on the testing phase. Interestingly, the microinjection of AM251 (20 and 40 ng/rat), but not WIN55,212-2 (0.1-0.5 μg/rat), into the BLA inhibited the expression of nicotine-induced place preference without any effect on the locomotor activity. Taken together, these findings support the possible role of endogenous cannabinoid system of the BLA in the acquisition and the expression of nicotine-induced place preference. Furthermore, it seems that there is a functional interaction between the BLA cannabinoid receptors and nicotine in producing the rewarding effects.

  9. Ouabain-induced perturbations in intracellular ionic homeostasis regulate death receptor-mediated apoptosis

    Science.gov (United States)

    Panayiotidis, Mihalis I.; Franco, Rodrigo; Bortner, Carl D.; Cidlowski, John A.

    2012-01-01

    Apoptosis is defined by specific morphological and biochemical characteristics including cell shrinkage (termed apoptotic volume decrease), a process that results from the regulation of ion channels and plasma membrane transporter activity. The Na+-K+-ATPase is the predominant pump that controls cell volume and plasma membrane potential in cells and alterations in its function have been suggested to be associated with apoptosis. We report here that the Na+-K+-ATPase inhibitor ouabain, potentiates apoptosis in the human lymphoma Jurkat cells exposed to Fas ligand (FasL) or Tumor necrosis factor--related apoptosis-inducing ligand (TRAIL) but not other apoptotic agents such as H2O2, thapsigargin or UV-C implicating a role for the Na+-K+-ATPase in death receptor-induced apoptosis. Interestingly, ouabain also potentiated perturbations in cell Ca2+ homeostasis only in conjunction with the apoptotic inducer FasL but not TRAIL. Ouabain did not affect alterations in the intracellular Ca2+ levels in response to H2O2, thapsigargin or UV-C. FasL-induced alterations in Ca2+ were not abolished in Ca2+-free medium but incubation of cells with BAPTA-AM inhibited both Ca2+ perturbations and the ouabain-induced potentiation of FasL-induced apoptosis. Our data suggest that the impairment of the Na+-K+-ATPase activity during apoptosis is linked to perturbations in cell Ca2+ homeostasis that modulate apoptosis induced by the activation of Fas by FasL. PMID:20422450

  10. P2X7 receptors mediate resistance to toxin-induced cell lysis.

    Science.gov (United States)

    Schoenauer, Roman; Atanassoff, Alexander P; Wolfmeier, Heidi; Pelegrin, Pablo; Babiychuk, Eduard B; Draeger, Annette

    2014-05-01

    In the majority of cells, the integrity of the plasmalemma is recurrently compromised by mechanical or chemical stress. Serum complement or bacterial pore-forming toxins can perforate the plasma membrane provoking uncontrolled Ca(2+) influx, loss of cytoplasmic constituents and cell lysis. Plasmalemmal blebbing has previously been shown to protect cells against bacterial pore-forming toxins. The activation of the P2X7 receptor (P2X7R), an ATP-gated trimeric membrane cation channel, triggers Ca(2+) influx and induces blebbing. We have investigated the role of the P2X7R as a regulator of plasmalemmal protection after toxin-induced membrane perforation caused by bacterial streptolysin O (SLO). Our results show that the expression and activation of the P2X7R furnishes cells with an increased chance of surviving attacks by SLO. This protective effect can be demonstrated not only in human embryonic kidney 293 (HEK) cells transfected with the P2X7R, but also in human mast cells (HMC-1), which express the receptor endogenously. In addition, this effect is abolished by treatment with blebbistatin or A-438079, a selective P2X7R antagonist. Thus blebbing, which is elicited by the ATP-mediated, paracrine activation of the P2X7R, is part of a cellular non-immune defense mechanism. It pre-empts plasmalemmal damage and promotes cellular survival. This mechanism is of considerable importance for cells of the immune system which carry the P2X7R and which are specifically exposed to toxin attacks.

  11. Therapeutic Implications of Progesterone Receptor-Mediated Regulation of Cell Cycle in Breast Cancer

    Science.gov (United States)

    2008-10-01

    surprise, we saw a biphasic dose response curve , with lower concentrations of R5020 (100 pM, or 10-10 M) inducing the most robust E2F1 expression at...a classic dose response curve , with maximal activation at the highest concentrations of R5020. However, R5020-mediated induction of E2F1 displays a...biphasic dose response curve ; lower concentrations of R5020 (100 pM, or 10-10 M) induce the most robust E2F1 expression, while higher

  12. Impaired P2X1 Receptor-Mediated Adhesion in Eosinophils from Asthmatic Patients.

    Science.gov (United States)

    Wright, Adam; Mahaut-Smith, Martyn; Symon, Fiona; Sylvius, Nicolas; Ran, Shaun; Bafadhel, Mona; Muessel, Michelle; Bradding, Peter; Wardlaw, Andrew; Vial, Catherine

    2016-06-15

    Eosinophils play an important role in the pathogenesis of asthma and can be activated by extracellular nucleotides released following cell damage or inflammation. For example, increased ATP concentrations were reported in bronchoalveolar lavage fluids of asthmatic patients. Although eosinophils are known to express several subtypes of P2 receptors for extracellular nucleotides, their function and contribution to asthma remain unclear. In this article, we show that transcripts for P2X1, P2X4, and P2X5 receptors were expressed in healthy and asthmatic eosinophils. The P2X receptor agonist α,β-methylene ATP (α,β-meATP; 10 μM) evoked rapidly activating and desensitizing inward currents (peak 18 ± 3 pA/pF at -60 mV) in healthy eosinophils, typical of P2X1 homomeric receptors, which were abolished by the selective P2X1 antagonist NF449 (1 μM) (3 ± 2 pA/pF). α,β-meATP-evoked currents were smaller in eosinophils from asthmatic patients (8 ± 2 versus 27 ± 5 pA/pF for healthy) but were enhanced following treatment with a high concentration of the nucleotidase apyrase (17 ± 5 pA/pF for 10 IU/ml and 11 ± 3 pA/pF for 0.32 IU/ml), indicating that the channels are partially desensitized by extracellular nucleotides. α,β-meATP (10 μM) increased the expression of CD11b activated form in eosinophils from healthy, but not asthmatic, donors (143 ± 21% and 108 ± 11% of control response, respectively). Furthermore, α,β-meATP increased healthy (18 ± 2% compared with control 10 ± 1%) but not asthmatic (13 ± 1% versus 10 ± 0% for control) eosinophil adhesion. Healthy human eosinophils express functional P2X1 receptors whose activation leads to eosinophil αMβ2 integrin-dependent adhesion. P2X1 responses are constitutively reduced in asthmatic compared with healthy eosinophils, probably as the result of an increase in extracellular nucleotide concentration.

  13. Effects of receptor-mediated endocytosis and tubular protein composition on volume retention in experimental glomerulonephritis

    DEFF Research Database (Denmark)

    Kastner, Christian; Pohl, Marcus; Sendeski, Mauricio

    2009-01-01

    Human glomerulonephritis (GN) is characterized by sustained proteinuria, sodium retention, hypertension, and edema formation. Increasing quantities of filtered protein enter the renal tubule, where they may alter epithelial transport functions. Exaggerated endocytosis and consequent protein overl...... mechanism of channel activation which may involve the action of filtered plasma proteases....... and channels involved in volume regulation were altered in GN, and 2) proximal tubular endocytosis may influence locally as well as downstream expressed tubular transporters and channels. Effects of anti-glomerular basement membrane GN were studied in controls and megalin-deficient mice with blunted proximal...

  14. The plasma concentration of HDL-associated apoM is influenced by LDL receptor-mediated clearance of apoB-containing particles

    DEFF Research Database (Denmark)

    Christoffersen, Christina; Benn, Marianne; Christensen, Pernille Møller;

    2012-01-01

    are affected by the rate of LDL receptor-mediated clearance of apoB-containing particles. We measured apoM in humans each carrying one of three different LDL receptor mutations (n = 9) or the apoB3500 mutation (n = 12). These carriers had increased plasma apoM (1.34 ± 0.13 µM, P = 0.003, and 1.23 ± 0.10 µM, P...... catabolic rate of LDL (r = -0.38, P = 0.009). These data suggest that the plasma clearance of apoM, despite apoM primarily being associated with HDL, is influenced by LDL receptor-mediated clearance of apoB-containing particles....

  15. Preparation and characterization of folate-poly(ethylene glycol)-grafted-trimethylchitosan for intracellular transport of protein through folate receptor-mediated endocytosis.

    Science.gov (United States)

    Zheng, Yu; Song, Xiangrong; Darby, Michael; Liang, Yufeng; He, Ling; Cai, Zheng; Chen, Qiuhong; Bi, Yueqi; Yang, Xiaojuan; Xu, Jiapeng; Li, Yuanbo; Sun, Yiyi; Lee, Robert J; Hou, Shixiang

    2010-01-01

    To develop a receptor-mediated intracellular delivery system that can transport therapeutic proteins to specific tumor cells, folate-poly(ethylene glycol)-grafted-trimethylchitosan (folate-PEG-g-TMC) was synthesized. Nano-scaled spherical polyelectrolyte complexes between the folate-PEG-g-TMC and fluorescein isothiocyanate conjugated bovine serum albumin (FITC-BSA) were prepared under suitable weight ratio of copolymer to FITC-BSA by ionic interaction between the positively charged copolymers and the negatively charged FITC-BSA. Intracellular uptake of FITC-BSA was specifically enhanced in SKOV3 cells (folate receptor over-expressing cell line) through folate receptor-mediated endocytosis compared with A549 cells (folate receptor deficient cell line). Folate-PEG-g-TMC shows promise for intracellular transport of negatively charged therapeutic proteins into folate receptor over-expressing tumor cells.

  16. Monoacylglycerol lipase promotes Fcγ receptor-mediated phagocytosis in microglia but does not regulate LPS-induced upregulation of inflammatory cytokines.

    Science.gov (United States)

    Kouchi, Zen

    2015-08-21

    Monoacylglycerol lipase (MAGL) is important for neuroinflammation. However, the regulatory mechanisms underlying its expression and function remain unknown. Lipopolysaccharide (LPS) treatment post-translationally upregulated MAGL expression, whereas it downregulated MAGL transcription through a Stat6-mediated mechanism in microglia. Neither MAGL knockdown nor JZL-184, a selective MAGL inhibitor, suppressed LPS-induced upregulation of inflammatory cytokines in microglia. Moreover, exogenous expression of MAGL in BV-2 microglial cell line, which lacks endogenous MAGL, did not promote the induction of inflammatory cytokines by LPS treatment. Interestingly, MAGL knockdown reduced Fcγ receptor-mediated phagocytosis in primary microglia, and introduction of MAGL into the BV-2 cells increased Fcγ receptor-mediated phagocytosis. Collectively, these results suggest that MAGL regulates phagocytosis, but not LPS-mediated cytokine induction in microglia.

  17. Chlorotoxin Fused to IgG-Fc Inhibits Glioblastoma Cell Motility via Receptor-Mediated Endocytosis

    Directory of Open Access Journals (Sweden)

    Tomonari Kasai

    2012-01-01

    Full Text Available Chlorotoxin is a 36-amino acid peptide derived from Leiurus quinquestriatus (scorpion venom, which has been shown to inhibit low-conductance chloride channels in colonic epithelial cells. Chlorotoxin also binds to matrix metalloproteinase-2 and other proteins on glioma cell surfaces. Glioma cells are considered to require the activation of matrix metalloproteinase-2 during invasion and migration. In this study, for targeting glioma, we designed two types of recombinant chlorotoxin fused to human IgG-Fcs with/without a hinge region. Chlorotoxin fused to IgG-Fcs was designed as a dimer of 60 kDa with a hinge region and a monomer of 30 kDa without a hinge region. The monomeric and dimeric forms of chlorotoxin inhibited cell proliferation at 300 nM and induced internalization in human glioma A172 cells. The monomer had a greater inhibitory effect than the dimer; therefore, monomeric chlorotoxin fused to IgG-Fc was multivalently displayed on the surface of bionanocapsules to develop a drug delivery system that targeted matrix metalloproteinase-2. The target-dependent internalization of bionanocapsules in A172 cells was observed when chlorotoxin was displayed on the bionanocapsules. This study indicates that chlorotoxin fused to IgG-Fcs could be useful for the active targeting of glioblastoma cells.

  18. GPR55 regulates cannabinoid 2 receptor-mediated responses in human neutrophils

    Institute of Scientific and Technical Information of China (English)

    Nariman A B Balenga; Maria Waldhoer; Elma Aflaki; Julia Kargl; Wolfgang Platzer; Ralf Schr(o)der; Stefanie Bl(a)ttermann; Evi Kostenis; Andrew J Brown; Akos Heinemann

    2011-01-01

    The directional migration of neutrophils towards inflammatory mediators,such as chemokines and cannabinoids,occurs via the activation of seven transmembrane G protein coupled receptors (7TM/GPCRs) and is a highly organized process.A crucial role for controlling neutrophil migration has been ascribed to the cannabinoid CB2 receptor (CB2R),but additional modulatory sites distinct from CB2R have recently been suggested to impact CB2R-mediated effector functions in neutrophils.Here,we provide evidence that the recently de-orphanized 7TM/GPCR GPR55potently modulates CB2R-mediated responses.We show that GPR55 is expressed in human blood neutrophils and its activation augments the migratory response towards the CB2R agonist 2-arachidonoylglycerol (2-AG),while inhibiting neutrophil degranulation and reactive oxygen species (ROS) production.Using HEK293 and HL60 cell lines,along with primary neutrophils,we show that GPR55 and CB2R interfere with each other's signaling pathways at the level of small GTPases,such as Rac2 and Cdc42.This ultimately leads to cellular polarization and efficient migration as well as abrogation of degranulation and ROS formation in neutrophils.Therefore,GPR55 limits the tissueinjuring inflammatory responses mediated by CB2R,while it synergizes with CB2R in recruiting neutrophils to sites of inflammation.

  19. GABA(A) receptor-mediated presynaptic inhibition on glutamatergic transmission.

    Science.gov (United States)

    Yamamoto, Sokatsu; Yoshimura, Megumu; Shin, Min-Chul; Wakita, Masahito; Nonaka, Kiku; Akaike, Norio

    2011-01-15

    We investigated the functional roles of presynaptic GABA(A) receptors on excitatory nerve terminals in contributing to spontaneous and action potential-evoked glutamatergic transmission to rat hippocampal CA3 pyramidal neurons. Single CA3 neurons were mechanically isolated with adherent nerve terminals, namely the 'synaptic bouton preparation', and spontaneous glutamatergic excitatory synaptic potentials (sEPSCs) and EPSCs evoked by focal electrical stimuli of a single presynaptic glutamatergic boutons (eEPSCs) were recorded using conventional whole-cell patch recordings. Selective activation of presynaptic GABA(A) receptors on these excitatory nerve terminals by muscimol, markedly facilitated sEPSCs frequency but inhibited eEPSC amplitude. The facilitation of sEPSC frequency was completely occluded by GABA(A) receptor-Cl⁻ channel blockers bicuculline or penicillin (PN). PN itself concentration-dependently inhibited the GABA(A) receptor response induced by bath application of muscimol, but had no effect on the glutamate receptor response. In addition, pretreatment with a blocker of the Na(+), K(+), 2Cl⁻ co-transporter type 1 (NKCC-1), bumetanide, prevented the muscimol-induced inhibition of eEPSCs. The results indicate that activation of presynaptic GABA(A) receptors directly depolarizes glutamatergic excitatory nerve terminals and thereby differentially modulates sEPSCs and eEPSCs.

  20. Ursodeoxycholic Acid Induces Death Receptor-mediated Apoptosis in Prostate Cancer Cells

    Science.gov (United States)

    Lee, Won Sup; Jung, Ji Hyun; Panchanathan, Radha; Yun, Jeong Won; Kim, Dong Hoon; Kim, Hye Jung; Kim, Gon Sup; Ryu, Chung Ho; Shin, Sung Chul; Hong, Soon Chan; Choi, Yung Hyun; Jung, Jin-Myung

    2017-01-01

    Background Bile acids have anti-cancer properties in a certain types of cancers. We determined anticancer activity and its underlying molecular mechanism of ursodeoxycholic acid (UDCA) in human DU145 prostate cancer cells. Methods Cell viability was measured with an MTT assay. UDCA-induced apoptosis was determined with flow cytometric analysis. The expression levels of apoptosis-related signaling proteins were examined with Western blotting. Results UDCA treatment significantly inhibited cell growth of DU145 in a dose-dependent manner. It induced cellular shrinkage and cytoplasmic blebs and accumulated the cells with sub-G1 DNA contents. Moreover, UDCA activated caspase 8, suggesting that UDCA-induced apoptosis is associated with extrinsic pathway. Consistent to this finding, UDCA increased the expressions of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor, death receptor 4 (DR4) and death receptor 5 (DR5), and TRAIL augmented the UDCA-induced cell death in DU145 cells. In addition, UDCA also increased the expressions of Bax and cytochrome c and decreased the expression of Bcl-xL in DU145 cells. This finding suggests that UDCA-induced apoptosis may be involved in intrinsic pathway. Conclusions UDCA induces apoptosis via extrinsic pathway as well as intrinsic pathway in DU145 prostate cancer cells. UDCA may be a promising anti-cancer agent against prostate cancer.

  1. Possible Implication of Fcγ Receptor-Mediated Trogocytosis in Susceptibility to Systemic Autoimmune Disease

    Directory of Open Access Journals (Sweden)

    Sakiko Masuda

    2013-01-01

    Full Text Available Leukocytes can “gnaw away” the plasma membrane of other cells. This phenomenon, called trogocytosis, occurs subsequent to cell-to-cell adhesion. Currently, two mechanisms of trogocytosis, adhesion molecule-mediated trogocytosis and Fcγ receptor-(FcγR- mediated trogocytosis, have been identified. In our earlier study, we established an in vitro model of FcγR-mediated trogocytosis, namely, CD8 translocation model from T cells to neutrophils. By using this model, we demonstrated that the molecules transferred to neutrophils via FcγR-mediated trogocytosis were taken into the cytoplasm immediately. This result suggests that the chance of molecules transferred via FcγR-mediated trogocytosis to play a role on the cell surface could be time-limited. Thus, we consider the physiological role of FcγR-mediated trogocytosis as a means to remove antibodies (Abs that bind with self-molecules rather than to extract molecules from other cells. This concept means that FcγR-mediated trogocytosis can be a defense mechanism to Ab-mediated autoimmune response. Moreover, the activity of FcγR-mediated trogocytosis was revealed to be parallel to the endocytotic activity of neutrophils, which was critically related to the susceptibility to systemic autoimmune diseases. The collective findings suggest that FcγR-mediated trogocytosis could physiologically play a role in removal of Abs bound to self-antigens and prevent autoimmune diseases.

  2. Nicotinic receptor mediates nitric oxide synthase expression in the rat gastric myenteric plexus.

    Science.gov (United States)

    Nakamura, K; Takahashi, T; Taniuchi, M; Hsu, C X; Owyang, C

    1998-04-01

    The mechanism that regulates the synthesis of nitric oxide synthase (NOS), a key enzyme responsible for NO production in the myenteric plexus, remains unknown. We investigated the roles of the vagal nerve and nicotinic synapses in the mediation of NOS synthesis in the gastric myenteric plexus in rats. Truncal vagotomy and administration of hexamethonium significantly reduced nonadrenergic, noncholinergic relaxation, the catalytic activity of NOS, the number of NOS-immunoreactive cells, and the density of NOS-immunoreactive bands and NOS mRNA bands obtained from gastric tissue. These results suggest that NOS expression in the gastric myenteric plexus is controlled by the vagal nerve and nicotinic synapses. We also investigated if stimulation of the nicotinic receptor increases neuronal NOS (nNOS) expression in cultured gastric myenteric ganglia. Incubation of cultured gastric myenteric ganglia with the nicotinic receptor agonist, 1,1-dimethyl-4-phenylpiperizinium (DMPP, 10(-10)-10(-7) M), for 24 h significantly increased the number of nNOS-immunoreactive cells and the density of immunoreactive nNOS bands and nNOS mRNA bands. nNOS mRNA expression stimulated by DMPP was antagonized by a protein kinase C antagonist, a phospholipase C inhibitor, and an intracellular Ca2+ chelator. We concluded that activation of the nicotinic receptor stimulates a Ca2+-dependent protein kinase C pathway, which in turn, upregulates nNOS mRNA expression and nNOS synthesis in the gastric myenteric plexus.

  3. Modulation of the leptin receptor mediates tumor growth and migration of pancreatic cancer cells.

    Science.gov (United States)

    Mendonsa, Alisha M; Chalfant, Madeleine C; Gorden, Lee D; VanSaun, Michael N

    2015-01-01

    Obesity has been implicated as a significant risk factor for development of pancreatic cancer. In the setting of obesity, a systemic chronic inflammatory response is characterized by alterations in the production and secretion of a wide variety of growth factors. Leptin is a hormone whose level increases drastically in the serum of obese patients. High fat diet induced obesity in mice leads to an overall increased body weight, pancreatic weight, serum leptin, and pancreatic tissue leptin levels. Here we report the contribution of obesity and leptin to pancreatic cancer growth utilizing an in vivo orthotopic murine pancreatic cancer model, which resulted in increased tumor proliferation with concomitant increased tumor burden in the diet induced obese mice compared to lean mice. Human and murine pancreatic cancer cell lines were found to express the short as well as the long form of the leptin receptor and functionally responded to leptin induced activation through an increased phosphorylation of AKT473. In vitro, leptin stimulation increased cellular migration which was blocked by addition of a PI3K inhibitor. In vivo, depletion of the leptin receptor through shRNA knockdown partially abrogated increased orthotopic tumor growth in obese mice. These findings suggest that leptin contributes to pancreatic tumor growth through activation of the PI3K/AKT pathway, which promotes pancreatic tumor cell migration.

  4. Modulation of the leptin receptor mediates tumor growth and migration of pancreatic cancer cells.

    Directory of Open Access Journals (Sweden)

    Alisha M Mendonsa

    Full Text Available Obesity has been implicated as a significant risk factor for development of pancreatic cancer. In the setting of obesity, a systemic chronic inflammatory response is characterized by alterations in the production and secretion of a wide variety of growth factors. Leptin is a hormone whose level increases drastically in the serum of obese patients. High fat diet induced obesity in mice leads to an overall increased body weight, pancreatic weight, serum leptin, and pancreatic tissue leptin levels. Here we report the contribution of obesity and leptin to pancreatic cancer growth utilizing an in vivo orthotopic murine pancreatic cancer model, which resulted in increased tumor proliferation with concomitant increased tumor burden in the diet induced obese mice compared to lean mice. Human and murine pancreatic cancer cell lines were found to express the short as well as the long form of the leptin receptor and functionally responded to leptin induced activation through an increased phosphorylation of AKT473. In vitro, leptin stimulation increased cellular migration which was blocked by addition of a PI3K inhibitor. In vivo, depletion of the leptin receptor through shRNA knockdown partially abrogated increased orthotopic tumor growth in obese mice. These findings suggest that leptin contributes to pancreatic tumor growth through activation of the PI3K/AKT pathway, which promotes pancreatic tumor cell migration.

  5. Fc-receptor-mediated phagocytosis is regulated by mechanical properties of the target

    Science.gov (United States)

    Beningo, Karen A.; Wang, Yu-li

    2002-01-01

    Phagocytosis is an actin-based process used by macrophages to clear particles greater than 0.5 microm in diameter. In addition to its role in immunological responses, phagocytosis is also necessary for tissue remodeling and repair. To prevent catastrophic autoimmune reactions, phagocytosis must be tightly regulated. It is commonly assumed that the recognition/selection of phagocytic targets is based solely upon receptor-ligand binding. Here we report an important new criterion, that mechanical parameters of the target can dramatically affect the efficiency of phagocytosis. When presented with particles of identical chemical properties but different rigidity, macrophages showed a strong preference to engulf rigid objects. Furthermore, phagocytosis of soft particles can be stimulated with the microinjection of constitutively active Rac1 but not RhoA, and with lysophosphatidic acid, an agent known to activate the small GTP-binding proteins of the Rho family. These data suggest a Rac1-dependent mechanosensory mechanism for phagocytosis, which probably plays an important role in a number of physiological and pathological processes from embryonic development to autoimmune diseases.

  6. Androgen Receptor-Mediated Genomic Androgen Action Augments Ischemia-Induced Neovascularization.

    Science.gov (United States)

    Lam, Yuen Ting; Lecce, Laura; Tan, Joanne T M; Bursill, Christina A; Handelsman, David J; Ng, Martin K C

    2016-12-01

    Increasing evidence indicates that androgens regulate ischemia-induced neovascularization. However, the role of genomic androgen action mediated by androgen receptor (AR), a ligand-activated nuclear transcription factor, remains poorly understood. Using an AR knockout (KO) mouse strain that contains a transcriptionally inactive AR (AR(Δex3)KO), we examined the role of AR genomic function in modulating androgen-mediated augmentation of ischemia-induced neovascularization. Castrated wild-type (AR(WT)) and AR(Δex3)KO mice were implanted with 5α-dihydrotestosterone (DHT) or placebo pellets after hindlimb ischemia (HLI). DHT modulation of angiogenesis and vasculogenesis, key processes for vascular repair and regeneration, was examined. Laser Doppler perfusion imaging revealed that DHT enhanced blood flow recovery in AR(WT) mice post-HLI. In AR(WT) mice, DHT enhanced angiogenesis by down-regulating prolyl hydroxylase 2 and augmenting hypoxia-inducible factor-1α (HIF-1α) levels in the ischemic tissues post-HLI. DHT also enhanced the production and mobilization of Sca1+/CXCR4+ progenitor cells in the bone marrow (BM) and circulating blood, respectively, in AR(WT) mice. By contrast, DHT-mediated enhancement of blood flow recovery was abrogated in AR(Δex3)KO mice. DHT modulation of HIF-1α expression was attenuated in AR(Δex3)KO mice. DHT-induced HIF-1α transcriptional activity and DHT-augmented paracrine-mediated endothelial cell tubule formation were attenuated in fibroblasts isolated from AR(Δex3)KO mice in vitro. Furthermore, DHT-induced augmentation of Sca1+/CXCR4+ progenitor cell production and mobilization was absent in AR(Δex3)KO mice post-HLI. BM transplantation revealed that ischemia-induced mobilization of circulating progenitor cells was abolished in recipients of AR(Δex3)KO BM. Together, these results indicate that androgen-mediated augmentation of ischemia-induced neovascularization is dependent on genomic AR transcriptional activation.

  7. Acute neuregulin-1 signaling influences AMPA receptor mediated responses in cultured cerebellar granule neurons.

    Science.gov (United States)

    Fenster, Catherine; Vullhorst, Detlef; Buonanno, Andres

    2012-01-04

    Neuregulin-1 (NRG1) is a trophic and differentiation factor that signals through ErbB receptor tyrosine kinases to regulate nervous system development. Previous studies have demonstrated that NRG1 affects plasticity at glutamatergic synapses in principal glutamatergic neurons of the hippocampus and frontal cortex; however, immunohistochemical and genetic analyses strongly suggest these effects are indirect and mediated via ErbB4 receptors on GABAergic interneurons. Here, we used cultured cerebellar granule cells (CGCs) that express ErbB4 to analyze the cell-autonomous effects of NRG1 stimulation on glutamatergic function. These cultures have the advantage that they are relatively homogenous and consist primarily of granule neurons that express ErbB4. We show that acute NRG1 treatment does not affect whole-cell AMPA or NMDA receptor (NMDAR) mediated currents in CGCs at 10-12 days in vitro. NRG1 also does not affect the frequency or amplitude of spontaneous AMPAR or NMDAR mediated miniature excitatory post-synaptic currents (mEPSCs). To further investigate the effects of NRG1 on activity-dependent plasticity of glutamatergic synapses in CGCs, we characterized the effects of high-glyine/0 Mg(2+) (which activates synaptic NMDARs) on AMPAR-mEPSC frequency and amplitude. We show that high-glycine induces a form of chemical long-term potentiation (chemLTP) in CGCs characterized by an increase in AMPAR-mEPSC frequency but not amplitude. Moreover, NRG1 induces a decrease in AMPAR-mEPSC frequency following chemLTP, but does not affect AMPAR-mEPSC amplitude. CGCs in our cultures conditions express low levels of GluR1, in contrast to dissociated hippocampal cultures, but do express the long isoform of GluR4. This study provides first evidence that (1) high-glycine can induce plasticity at glutamatergic synapses in CGCs, and (2) that acute NRG1/ErbB-signaling can regulate glutamatergic plasticity in CGCs. Taken together with previous reports, our results suggest that, similar

  8. Subcellular localization of frizzled receptors, mediated by their cytoplasmic tails, regulates signaling pathway specificity.

    Directory of Open Access Journals (Sweden)

    Jun Wu

    2004-07-01

    Full Text Available The Frizzled (Fz; called here Fz1 and Fz2 receptors have distinct signaling specificities activating either the canonical Wnt/beta-catenin pathway or Fz/planar cell polarity (PCP signaling in Drosophila. The regulation of signaling specificity remains largely obscure. We show that Fz1 and Fz2 have different subcellular localizations in imaginal disc epithelia, with Fz1 localizing preferentially to apical junctional complexes, and Fz2 being evenly distributed basolaterally. The subcellular localization difference directly contributes to the signaling specificity outcome. Whereas apical localization favors Fz/PCP signaling, it interferes with canonical Wnt/beta-catenin signaling. Receptor localization is mediated by sequences in the cytoplasmic tail of Fz2 that appear to block apical accumulation. Based on these data, we propose that subcellular Fz localization, through the association with other membrane proteins, is a critical aspect in regulating the signaling specificity within the Wnt/Fz signaling pathways.

  9. Cholinergic impact on neuroplasticity drives muscarinic M1 receptor mediated differentiation into neurons.

    Science.gov (United States)

    Benninghoff, Jens; Rauh, Werner; Brantl, Victor; Schloesser, Robert J; Moessner, Rainald; Möller, Hans-Jürgen; Rujescu, Dan

    2013-04-01

    Increasing evidence indicates that canonical neurotransmitters act as regulatory signals during neuroplasticity. Here, we report that muscarinic cholinergic neurotransmission stimulates differentiation of adult neural stem cells in vitro. Adult neural stem cells (ANSC) dissociated from the adult mouse hippocampus were expanded in culture with basic fibroblast growth factor (BFGF) and epidermal growth factor (EGF). Carbachol (CCh), an analog of acetylcholine (ACh) significantly enhanced de novo differentiation into neurons on bFGF- and EGF-deprived stem cells as shown by the percentage of TUJ1 positive cells. By contrast, pirenzepine (PIR), a muscarinic M1 receptor antagonist, reduced the generation of neurons. Activation of cholinergic signaling drives the de novo differentiation of uncommitted stem cells into neurons. These effects appear to be predominantly mediated via the muscarinic M1 receptor subtype.

  10. Downregulation of adenosine and P2X receptor-mediated cardiovascular responses in heart failure rats

    DEFF Research Database (Denmark)

    Zhao, Xin; Sun, X Y; Erlinge, D;

    2000-01-01

    Neurohormonal changes in congestive heart failure (CHF) include an enhanced peripheral sympathetic nerve activity which results in increased release of noradrenaline, neuropeptide Y and ATP. To examine if such changes in CHF would modulate peripheral pre- and postsynaptic receptors of ATP and its...... effects mediated by the endothelial P2Y receptors are unaffected in CHF. Moreover, the adenosine-mediated inhibitory effects on heart rate and blood pressure were also attenuated in the CHF rats. The most important changes in adenosine and P2-receptor function induced by ischaemic CHF were the reduced...... pressor effect mediated by the P2X receptor and the increased heart rate due to an attenuated inhibitory effect of adenosine....

  11. The CRF family of neuropeptides and their receptors - mediators of the central stress response.

    Science.gov (United States)

    Dedic, Nina; Chen, Alon; Deussing, Jan M

    2017-03-01

    Dysregulated stress neurocircuits, caused by genetic and/or environmental changes, underlie the development of many neuropsychiatric disorders. Corticotropin-releasing factor (CRF) is the major physiological activator of the hypothalamic-pituitary-adrenal (HPA) axis and consequently a primary regulator of the mammalian stress response. Together with its three family members, urocortins (UCNs) 1, 2, and 3, CRF integrates the neuroendocrine, autonomic, metabolic and behavioral responses to stress by activating its cognate receptors CRFR1 and CRFR2. Here we review the past and current state of the CRH/CRHR field, ranging from pharmacological studies to genetic mouse models and virus-mediated manipulations. Although it is well established that CRF/CRFR1 signaling mediates aversive responses, including anxiety and depression-like behaviors, a number of recent studies have challenged this viewpoint by revealing anxiolytic and appetitive properties of specific CRF/CRFR1 circuits. In contrast, the UCN/CRFR2 system is less well understood and may possibly also exert divergent functions on physiology and behavior depending on the brain region,underlying circuit, and/or experienced stress conditions. A plethora of available genetic tools, including conventional and conditional mouse mutants targeting CRF system components, has greatly advanced our understanding about the endogenous mechanisms underlying HPA system regulation and CRH/UCN-related neuronal circuits involved in stress-related behaviors. Yet, the deailed pathways and molecular mechanism by which the CRH/UCN-system translates negative or positive stimuli into the final, integrated biological response are not completely understood. The utilization of future complementary methodologies, such as cell-type specific Cre-driver lines, viral and optogenetic tools will help to further dissect the function of genetically defined CRH/UCN neurocircuits in the context of adaptive and maladaptive stress responses. Copyright

  12. Fcgamma receptor-mediated suppression of human immunodeficiency virus type 1 replication in primary human macrophages.

    Science.gov (United States)

    Perez-Bercoff, Danielle; David, Annie; Sudry, Hugues; Barré-Sinoussi, Françoise; Pancino, Gianfranco

    2003-04-01

    Permissiveness of monocytes and macrophages to human immunodeficiency virus (HIV) infection is modulated by various stimuli. In this study we demonstrate that stimulation of primary monocytes and monocyte-derived macrophages (MDM) through the receptors for the Fc portion of immunoglobulin G (IgG) (FcgammaR) inhibits HIV type 1 (HIV-1) replication. Viral p24 production was decreased by 1.5 to 3 log units in MDM infected with both R5 and X4 HIV-1 strains upon stimulation by immobilized IgG but not upon stimulation by soluble IgG or by F(ab')(2) IgG fragments. Although MDM activation by immobilized IgG induced high levels of macrophage-derived chemokine secretion as well as a sustained down-regulation of CD4 and a transient decrease in CCR5 expression, these factors did not appear to play a major role in the suppression of HIV-1 replication. Single-cycle infection of FcgammaR-stimulated MDM with HIV-1 virions pseudotyped with either HIV-1 R5 or vesicular stomatitis virus G envelopes was inhibited, suggesting a postentry restriction of viral replication. PCR analyses of HIV-1 DNA intermediate replication forms suggested that reverse transcription is not affected by stimulation with immobilized human IgG, at least during the first replication cycle. The accumulation of PCR products corresponding to nuclear unintegrated two-long-terminal-repeat circles and the relative decrease of integrated HIV-1 DNA signals suggest an inhibition of proviral integration. Our data, showing that FcgammaR-mediated activation of MDM is a potent mechanism of HIV-1 suppression, raise the possibility that FcgammaR cross-linking by immune complexes may contribute to the control of viral replication in macrophages.

  13. Angiotensin II Type 1 Receptor-Mediated Electrical Remodeling in Mouse Cardiac Myocytes.

    Directory of Open Access Journals (Sweden)

    Jeremy Kim

    Full Text Available We recently characterized an autocrine renin angiotensin system (RAS in canine heart. Activation of Angiotensin II Type 1 Receptors (AT1Rs induced electrical remodeling, including inhibition of the transient outward potassium current Ito, prolongation of the action potential (AP, increased calcium entry and increased contractility. Electrical properties of the mouse heart are very different from those of dog heart, but if a similar system existed in mouse, it could be uniquely studied through genetic manipulations. To investigate the presence of a RAS in mouse, we measured APs and Ito in isolated myocytes. Application of angiotensin II (A2 for 2 or more hours reduced Ito magnitude, without affecting voltage dependence, and prolonged APs in a dose-dependent manner. Based on dose-inhibition curves, the fast and slow components of Ito (Ito,fast and IK,slow appeared to be coherently regulated by [A2], with 50% inhibition at an A2 concentration of about 400 nM. This very high K0.5 is inconsistent with systemic A2 effects, but is consistent with an autocrine RAS in mouse heart. Pre-application of the microtubule destabilizing agent colchicine eliminated A2 effects on Ito and AP duration, suggesting these effects depend on intracellular trafficking. Application of the biased agonist SII ([Sar1-Ile4-Ile8]A2, which stimulates receptor internalization without G protein activation, caused Ito reduction and AP prolongation similar to A2-induced changes. These data demonstrate AT1R mediated regulation of Ito in mouse heart. Moreover, all measured properties parallel those measured in dog heart, suggesting an autocrine RAS may be a fundamental feedback system that is present across species.

  14. Differential T cell receptor-mediated signaling in naive and memory CD4 T cells.

    Science.gov (United States)

    Farber, D L; Acuto, O; Bottomly, K

    1997-08-01

    Naive and memory CD4 T cells differ in cell surface phenotype, function, activation requirements, and modes of regulation. To investigate the molecular bases for the dichotomies between naive and memory CD4 T cells and to understand how the T cell receptor (TCR) directs diverse functional outcomes, we investigated proximal signaling events triggered through the TCR/CD3 complex in naive and memory CD4 T cell subsets isolated on the basis of CD45 isoform expression. Naive CD4 T cells signal through TCR/CD3 similar to unseparated CD4 T cells, producing multiple tyrosine-phosphorylated protein species overall and phosphorylating the T cell-specific ZAP-70 tyrosine kinase which is recruited to the CD3zeta subunit of the TCR. Memory CD4 T cells, however, exhibit a unique pattern of signaling through TCR/CD3. Following stimulation through TCR/CD3, memory CD4 T cells produce fewer species of tyrosine-phosphorylated substrates and fail to phosphorylate ZAP-70, yet unphosphorylated ZAP-70 can associate with the TCR/CD3 complex. Moreover, a 26/28-kDa phosphorylated doublet is associated with CD3zeta in resting and activated memory but not in naive CD4 T cells. Despite these differences in the phosphorylation of ZAP-70 and CD3-associated proteins, the ZAP-70-related kinase, p72syk, exhibits similar phosphorylation in naive and memory T cell subsets, suggesting that this kinase could function in place of ZAP-70 in memory CD4 T cells. These results indicate that proximal signals are differentially coupled to the TCR in naive versus memory CD4 T cells, potentially leading to distinct downstream signaling events and ultimately to the diverse functions elicited by these two CD4 T cell subsets.

  15. Adolescent chronic mild stress alters hippocampal CB1 receptor-mediated excitatory neurotransmission and plasticity.

    Science.gov (United States)

    Reich, C G; Mihalik, G R; Iskander, A N; Seckler, J C; Weiss, M S

    2013-12-03

    Endocannabinoids (eCBs) are involved in the stress response and alterations in eCB signaling may contribute to the etiology of mood disorders. Exposure to chronic mild stress (CMS), a model of depression, produces downregulation of the cannabinoid 1 (CB1) receptor in the hippocampus of male rats. However, it is unknown how this stress-induced change in CB1 levels affects eCB-mediated neurotransmission. In vitro, field potential recordings from CMS-exposed (21-days) rats were performed to assess the effects of stress on eCB-regulated glutamatergic neurotransmission in/on hippocampal area CA1. We observed that application of the CB1 agonist, WIN 55,212-5 (1 μM), in stress animals resulted in a ∼135% increase in excitatory neurotransmission, whereas CB1 activation in non-stress animals leads to a ∼30% decrease. However, during blockade of GABA(A) neurotransmission with picrotoxin, CB1 activation yielded a ∼35% decrease in stress animals. These findings indicate that CMS does not directly affect glutamatergic neurotransmission. Rather, CMS sensitizes CB1 function on GABAergic terminals, leading to less inhibition and an increase in excitatory neurotransmission. This finding is reinforced in that induction of weak long-term-potentiation (LTP) is enhanced in CMS-exposed animals compared to controls and this enhancement is CB1-dependent. Lastly, we observed that the LTP-blocking property of WIN 55,212-5 shifts from being glutamate-dependent in non-stress animals to being GABA-dependent in stress animals. These results effectively demonstrate that CMS significantly alters hippocampal eCB-mediated neurotransmission and synaptic plasticity.

  16. A model for modulation of neuronal synchronization by D4 dopamine receptor-mediated phospholipid methylation.

    Science.gov (United States)

    Kuznetsova, Anna Y; Deth, Richard C

    2008-06-01

    We describe a new molecular mechanism of dopamine-induced membrane protein modulation that can tune neuronal oscillation frequency to attention-related gamma rhythm. This mechanism is based on the unique ability of D4 dopamine receptors (D4R) to carry out phospholipid methylation (PLM) that may affect the kinetics of ion channels. We show that by deceasing the inertia of the delayed rectifier potassium channel, a transition to 40 Hz oscillations can be achieved. Decreased potassium channel inertia shortens spike duration and decreases the interspike interval via its influence on the calcium-dependent potassium current. This mechanism leads to a transition to attention-related gamma oscillations in a pyramidal cell-interneuron network. The higher frequency and better synchronization is observed with PLM affecting pyramidal neurons only, and recurrent excitation between pyramidal neurons is important for synchronization. Thus dopamine-stimulated methylation of membrane phospholipids may be an important mechanism for modulating firing activity, while impaired methylation can contribute to disorders of attention.

  17. Cannabinoid CB2 receptor-mediated anti-nociception in models of acute and chronic pain.

    Science.gov (United States)

    Jhaveri, Maulik D; Sagar, Devi R; Elmes, Steven J R; Kendall, David A; Chapman, Victoria

    2007-08-01

    The endocannabinoid system consists of cannabinoid CB(1) and CB(2) receptors, endogenous ligands and their synthesising/metabolising enzymes. Cannabinoid receptors are present at key sites involved in the relay and modulation of nociceptive information. The analgesic effects of cannabinoids have been well documented. The usefulness of nonselective cannabinoid agonists can, however, be limited by psychoactive side effects associated with activation of CB(1) receptors. Following the recent evidence for CB(2) receptors existing in the nervous system and reports of their up-regulation in chronic pain states and neurodegenerative diseases, much research is now aimed at shedding light on the role of the CB(2) receptor in human disease. Recent studies have demonstrated anti-nociceptive effects of selective CB(2) receptor agonists in animal models of pain in the absence of CNS side effects. This review focuses on the analgesic potential of CB(2) receptor agonists for inflammatory, post-operative and neuropathic pain states and discusses their possible sites and mechanisms of action.

  18. Identification of a novel modulator of thyroid hormone receptor-mediated action.

    Directory of Open Access Journals (Sweden)

    Bernhard G Baumgartner

    Full Text Available BACKGROUND: Diabetes is characterized by reduced thyroid function and altered myogenesis after muscle injury. Here we identify a novel component of thyroid hormone action that is repressed in diabetic rat muscle. METHODOLOGY/PRINCIPAL FINDINGS: We have identified a gene, named DOR, abundantly expressed in insulin-sensitive tissues such as skeletal muscle and heart, whose expression is highly repressed in muscle from obese diabetic rats. DOR expression is up-regulated during muscle differentiation and its loss-of-function has a negative impact on gene expression programmes linked to myogenesis or driven by thyroid hormones. In agreement with this, DOR enhances the transcriptional activity of the thyroid hormone receptor TR(alpha1. This function is driven by the N-terminal part of the protein. Moreover, DOR physically interacts with TR( alpha1 and to T(3-responsive promoters, as shown by ChIP assays. T(3 stimulation also promotes the mobilization of DOR from its localization in nuclear PML bodies, thereby indicating that its nuclear localization and cellular function may be related. CONCLUSIONS/SIGNIFICANCE: Our data indicate that DOR modulates thyroid hormone function and controls myogenesis. DOR expression is down-regulated in skeletal muscle in diabetes. This finding may be of relevance for the alterations in muscle function associated with this disease.

  19. Role of NMDA Receptor-Mediated Glutamatergic Signaling in Chronic and Acute Neuropathologies

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    Francisco J. Carvajal

    2016-01-01

    Full Text Available N-Methyl-D-aspartate receptors (NMDARs have two opposing roles in the brain. On the one hand, NMDARs control critical events in the formation and development of synaptic organization and synaptic plasticity. On the other hand, the overactivation of NMDARs can promote neuronal death in neuropathological conditions. Ca2+ influx acts as a primary modulator after NMDAR channel activation. An imbalance in Ca2+ homeostasis is associated with several neurological diseases including schizophrenia, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis. These chronic conditions have a lengthy progression depending on internal and external factors. External factors such as acute episodes of brain damage are associated with an earlier onset of several of these chronic mental conditions. Here, we will review some of the current evidence of how traumatic brain injury can hasten the onset of several neurological conditions, focusing on the role of NMDAR distribution and the functional consequences in calcium homeostasis associated with synaptic dysfunction and neuronal death present in this group of chronic diseases.

  20. Metabotropic glutamate receptor-mediated long-term depression: molecular mechanisms.

    Science.gov (United States)

    Gladding, Clare M; Fitzjohn, Stephen M; Molnár, Elek

    2009-12-01

    The ability to modify synaptic transmission between neurons is a fundamental process of the nervous system that is involved in development, learning, and disease. Thus, synaptic plasticity is the ability to bidirectionally modify transmission, where long-term potentiation and long-term depression (LTD) represent the best characterized forms of plasticity. In the hippocampus, two main forms of LTD coexist that are mediated by activation of either N-methyl-d-aspartic acid receptors (NMDARs) or metabotropic glutamate receptors (mGluRs). Compared with NMDAR-LTD, mGluR-LTD is less well understood, but recent advances have started to delineate the underlying mechanisms. mGluR-LTD at CA3:CA1 synapses in the hippocampus can be induced either by synaptic stimulation or by bath application of the group I selective agonist (R,S)-3,5-dihydroxyphenylglycine. Multiple signaling mechanisms have been implicated in mGluR-LTD, illustrating the complexity of this form of plasticity. This review provides an overview of recent studies investigating the molecular mechanisms underlying hippocampal mGluR-LTD. It highlights the role of key molecular components and signaling pathways that are involved in the induction and expression of mGluR-LTD and considers how the different signaling pathways may work together to elicit a persistent reduction in synaptic transmission.

  1. Hindbrain GLP-1 receptor mediation of cisplatin-induced anorexia and nausea.

    Science.gov (United States)

    De Jonghe, Bart C; Holland, Ruby A; Olivos, Diana R; Rupprecht, Laura E; Kanoski, Scott E; Hayes, Matthew R

    2016-01-01

    While chemotherapy-induced nausea and vomiting are clinically controlled in the acute (anorexia, nausea, fatigue, and other illness-type behaviors during the delayed phase (>24 h) of chemotherapy are largely uncontrolled. As the hindbrain glucagon-like peptide-1 (GLP-1) system contributes to energy balance and mediates aversive and stressful stimuli, here we examine the hypothesis that hindbrain GLP-1 signaling mediates aspects of chemotherapy-induced nausea and reductions in feeding behavior in rats. Specifically, hindbrain GLP-1 receptor (GLP-1R) blockade, via 4th intracerebroventricular (ICV) exendin-(9-39) injections, attenuates the anorexia, body weight reduction, and pica (nausea-induced ingestion of kaolin clay) elicited by cisplatin chemotherapy during the delayed phase (48 h) of chemotherapy-induced nausea. Additionally, the present data provide evidence that the central GLP-1-producing preproglucagon neurons in the nucleus tractus solitarius (NTS) of the caudal brainstem are activated by cisplatin during the delayed phase of chemotherapy-induced nausea, as cisplatin led to a significant increase in c-Fos immunoreactivity in NTS GLP-1-immunoreactive neurons. These data support a growing body of literature suggesting that the central GLP-1 system may be a potential pharmaceutical target for adjunct anti-emetics used to treat the delayed-phase of nausea and emesis, anorexia, and body weight loss that accompany chemotherapy treatments.

  2. Metabotropic glutamate receptor-mediated signaling dampens the HPA axis response to restraint stress.

    Science.gov (United States)

    Evanson, Nathan K; Herman, James P

    2015-10-15

    Glutamate is an important neurotransmitter in the regulation of the neural portion of hypothalamus-pituitary-adrenal (HPA) axis activity, and signals through ionotropic and metabotropic receptors. In the current studies we investigated the role of hypothalamic paraventricular group I metabotropic glutamate receptors in the regulation of the HPA axis response to restraint stress in rats. Direct injection of the group I metabotropic glutamate receptor agonist 3,5-dihydroxyphenylglycine (DHPG) into the PVN prior to restraint leads to blunting of the HPA axis response in awake animals. Consistent with this result, infusion of the group I receptor antagonist hexyl-homoibotenic acid (HIBO) potentiates the HPA axis response to restraint. The excitatory effect of blocking paraventricular group I metabotropic glutamate signaling is blocked by co-administration of dexamethasone into the PVN. However, the inhibitory effect of DHPG is not affected by co-administration of the cannabinoid CB1 receptor antagonist AM-251 into the PVN. Together, these results suggest that paraventricular group I metabotropic glutamate receptor signaling acts to dampen HPA axis reactivity. This effect appears to be similar to the rapid inhibitory effect of glucocorticoids at the PVN, but is not mediated by endocannabinoid signaling.

  3. Benzodiazepine receptor-mediated behavioral effects of nitrous oxide in the rat social interaction test.

    Science.gov (United States)

    Quock, R M; Wetzel, P J; Maillefer, R H; Hodges, B L; Curtis, B A; Czech, D A

    1993-09-01

    The present study was conducted to ascertain whether an anxiolytic effect of nitrous oxide was demonstrable in rats using the social interaction test and whether this drug effect might be mediated by benzodiazepine receptors. Compared to behavior of vehicle-pretreated, room air-exposed rats, rat pairs exposed to nitrous oxide showed a generally inverted U-shaped dose-response curve with the maximum increase in social interaction encounters occurring at 25% and significant increase in time of active social interaction at 15-35%; higher concentrations produced a sedative effect that reduced social interaction. Treatment with 5.0 mg/kg of the anxiolytic benzodiazepine chlordiazepoxide also increased social interaction. Pretreatment with 10 mg/kg of the benzodiazepine receptor blocker flumazenil, which alone had no effect, significantly antagonized the social interaction-increasing effects of both nitrous oxide and chlordiazepoxide. In summary, these findings suggest that nitrous oxide produces a flumazenil-sensitive effect comparable to that of chlordiazepoxide and implicate central benzodiazepine mechanisms in mediation of the anxiolytic effect of nitrous oxide.

  4. T Cell Receptor Mediated Calcium Entry Requires Alternatively Spliced Cav1.1 Channels.

    Directory of Open Access Journals (Sweden)

    Didi Matza

    Full Text Available The process of calcium entry in T cells is a multichannel and multi-step process. We have studied the requirement for L-type calcium channels (Cav1.1 α1S subunits during calcium entry after TCR stimulation. High expression levels of Cav1.1 channels were detected in activated T cells. Sequencing and cloning of Cav1.1 channel cDNA from T cells revealed that a single splice variant is expressed. This variant lacks exon 29, which encodes the linker region adjacent to the voltage sensor, but contains five new N-terminal exons that substitute for exons 1 and 2, which are found in the Cav1.1 muscle counterpart. Overexpression studies using cloned T cell Cav1.1 in 293HEK cells (that lack TCR suggest that the gating of these channels was altered. Knockdown of Cav1.1 channels in T cells abrogated calcium entry after TCR stimulation, suggesting that Cav1.1 channels are controlled by TCR signaling.

  5. Receptor-mediated endocytosis of carcinoembryonic antigen by rat liver Kupffer cells.

    Science.gov (United States)

    Toth, C A; Thomas, P; Broitman, S A; Zamcheck, N

    1985-01-01

    In vivo, carcinoembryonic antigen (CEA) is removed from the circulation by the liver Kupffer cells. Immunologically identifiable CEA is transferred from these macrophages to the hepatocytes, where degradation is completed. Circulatory clearance of CEA is specific, rapid [t1/2 = 3.7 +/- 0.9 (S.D.) min], and saturable. In vitro, Kupffer cells take up CEA by a saturable process which is time/temperature dependent and colchicine sensitive. Isolated Kupffer cells endocytose CEA with an apparent Km of 6 X 10(-8) M. There are approximately 16,000 CEA binding sites per cell. Nonspecific cross-reacting antigen (NCA), a glycoprotein structurally similar to CEA, is recognized with lower affinity by the same receptor. Endocytosis is independent of the nonreducing terminal sugars on the molecule: CEA modified by Smith degradation inhibits Kupffer cell recognition of native CEA. Since performic acid oxidized CEA also inhibits endocytosis, receptor binding is similarly independent of intact protein conformation. Isolated Kupffer cells have mannose and/or N-acetyl glucosamine receptor activity but do not internalize CEA by that mechanism. Galactose-terminated glycoproteins impede CEA and NCA clearance in vivo but not Kupffer cell endocytosis in vitro. Radiolabeled CEA released from isolated Kupffer cells following endocytosis shows no apparent molecular weight change. However, the released CEA contains species with higher isoelectric points, suggesting that perhaps the removal of sialic acid and the resulting exposure of galactose residues mediate the subsequent transfer to the hepatocyte.

  6. beta. -adrenergic receptor-mediated hepatic glycogenolysis is increased in aged male rats

    Energy Technology Data Exchange (ETDEWEB)

    Herring, P.A.; Graham, S.M.; Arinze, I.J.

    1986-03-05

    The effect of age on catecholamine-stimulated glycogenolysis was studied in isolated hepatocytes prepared from 3, 12, and 24 month-old rats. Glucose release was stimulated by epinephrine and norepinephrine, this was inhibited by phentolamine and prazosin. Isoproterenol (ISO) stimulated glycogenolysis only in cells from 24 month-old rats, this was blocked by propranolol. In liver plasma membranes, binding of (/sup 3/H)yohimbine (100-130 fmol/mg protein) did not change with age, whereas (/sup 3/H)prazosin binding decreased from 870 fmol/mg at 3 months to 435 fmol/mg at 12 months, but subsequently rose to 656 fmol/mg at 24 months. (/sup 125/I)Cyanopindolol binding increased from 8 fmol/mg at 3 months to 19 fmol/mg at 24 months. The proportion of ..beta..-receptors in the high affinity state increased from 28% at 3 months to 42% at 24 months. ISO stimulated adenylate cyclase at 24 months but not at 3 months. Basal, fluoride-, GTP-, and Gpp(NH)p-stimulated activities were 1.4- to 2.4-fold greater at 24 months than at 3 months. These results suggest an age-related increase in the sensitivity of adenylate cyclase to ..beta..-receptor stimulation.

  7. P2X7 Receptor Mediated Growth-Inhibitory Effect in KG1 a Cell Line

    Institute of Scientific and Technical Information of China (English)

    Xiujun Zhang; Lijun Meng

    2008-01-01

    This study was conducted to investigate ATP-induced growth inhibition in human leukemic cells KGla.METHoDS ATP inhibited cell growth was analyzed by MTSassay.Extemalization of phosphatidylserine could be detected byAnnexin-V-FITC apoptosis staining after activation of the P2X7 re-ceptor.P2X7 mediated pore formation was detected in KGla cellsby Yo-Pro-1 uptake assay.RESUlTS ATP inhibited cell growth in a dose-dependent man-ner.The cytotoxic effect could be blocked by P2X7 antagonists,oxidized ATP(OATP)and KN62.Externalization of phosphatidyl-serine could be detected in a time-dependent manner.P2X7 medi-ated pore forigation could be detected in KGla cells.These effectscould not be observed in P2X7 null Ramos cells.CONCLUSIoN The results and our previously reports thatmRNA,protein expression and calcium response of the P2X7receptor in KGla cells,suggested that extracellular ATP effectivelyinduces growth inhibition through apoptosis in KGla cells byactivation of P2X7 receptor,and that may be mediated by extracel-lular Ca2+ineux and pore formation.

  8. NMDA and AMPA receptors mediate intracellular calcium increase in rat cortical astrocytes

    Institute of Scientific and Technical Information of China (English)

    Bo HU; Sheng-gang SUN; E-tang TONG

    2004-01-01

    AIM: To study the effect of glutamate on the intracellular calcium signal of pure cultured rat astrocytes and the role of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors in the procedure. METHODS: The fluorescence of calcium was measured by Fura-2/AM (F345/F380).RESULTS: L-Glutamate induced [Ca2+]i increase in most of the cells in concentration- and time-dependent manner.NMDA 50 mmol/L induced the fluorescence increase by almost three to four times, while the effect of AMPA 50mmol/L was just half of that of D-(-)-2-amino-5-phosphonopentanoic acid (D-AP-5; a selective antagonist of the NMDA receptor). 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX, a selective antagonist of the AMPA receptor)abolished the effects of NMDA and AMPA, respectively. D-AP-5 and CNQX simultaneously or respectively attenuated the effect of L-glutamate at different degrees, but could not abolish it entirely. CONCLUSION: Glutamate modulated intracellular Ca2+ of pure cultured rat astrocytes through different pathways. The activation of NMDA and AMPA receptors took part in the complex mechanisms.

  9. Neuroprotection against hypoxia/ischemia: δ-opioid receptor-mediated cellular/molecular events.

    Science.gov (United States)

    He, Xiaozhou; Sandhu, Harleen K; Yang, Yilin; Hua, Fei; Belser, Nathalee; Kim, Dong H; Xia, Ying

    2013-07-01

    Hypoxic/ischemic injury remains the most dreaded cause of neurological disability and mortality. Despite the humbling experiences due to lack of promising therapy, our understanding of the complex cascades underlying the neuronal insult has led to advances in basic science research. One of the most noteworthy has been the effect of opioid receptors, especially the delta-opioid receptor (DOR), on hypoxic/ischemic neurons. Our recent studies, and those of others worldwide, present strong evidence that sheds light on DOR-mediated neuroprotection in the brain, especially in the cortex. The mechanisms of DOR neuroprotection are broadly categorized as: (1) stabilization of the ionic homeostasis, (2) inhibition of excitatory transmitter release, (3) attenuation of disrupted neuronal transmission, (4) increase in antioxidant capacity, (5) regulation of intracellular pathways-inhibition of apoptotic signals and activation of pro-survival signaling, (6) regulation of specific gene and protein expression, and (7) up-regulation of endogenous opioid release and/or DOR expression. Depending upon the severity and duration of hypoxic/ischemic insult, the release of endogenous opioids and DOR expression are regulated in response to the stress, and DOR signaling acts at multiple levels to confer neuronal tolerance to harmful insult. The phenomenon of DOR neuroprotection offers a potential clue for a promising target that may have significant clinical implications in our quest for neurotherapeutics.

  10. T cell proliferative responses to malondialdehyde-acetaldehyde haptenated protein are scavenger receptor mediated.

    Science.gov (United States)

    Willis, Monte S; Thiele, Geoffrey M; Tuma, Dean J; Klassen, Lynell W

    2003-10-01

    Malondialdehyde-acetaldehyde (MAA) haptenated proteins have been described in disease processes related to prolonged oxidative stress (via malondialdehyde production), such as alcohol liver disease (ALD), non-alcoholic non-steatohepatitis (NASH) and atherosclerosis. Experimentally, high titer IgG1 antibody responses are seen after immunization without adjuvant; however, T cell proliferative responses and the role of scavenger receptors in this immunogenicity has not previously been described. In this study, T cell proliferative responses to the carrier protein, but not the MAA hapten itself, were identified in vitro. Moreover, these T proliferative responses were inhibited when MAA-hen egg lysozyme (HEL) was co-immunized with excess scavenger receptor ligand polyG (poly-guanylic acid), implicating the role of (a) scavenger receptor(s) in initiating the T helper cell response. Activated B cells were unable to process and present MAA-HEL preferentially to T cells, while thioglycollate-elicited (but not Con A-elicited) macrophages and dendritic cells (DC) did so with approximately 32-fold less MAA-HEL than native antigen necessary to initiate equal proliferative responses. While this preferential processing and presentation may be related to several factors, preferential binding of MAA haptenated proteins mediated by scavenger receptors may be one mechanism. IL-4 was absent from the supernatants of T proliferative assays despite a strong IgG1 response in vivo, although the TH2 cytokines IL-6 and IL-10 were expressed. Since the modification of proteins by the MAA have previously been shown to occur after ethanol consumption in vivo, the ability of MAA haptens to experimentally enhance immune responses, specifically humoral and T cell responses, may represent mechanisms by which autoimmune phenomena found in ALD occur.

  11. Muscarinic acetylcholine receptor-mediated effects in slices from human epileptogenic cortex.

    Science.gov (United States)

    Gigout, S; Wierschke, S; Lehmann, T-N; Horn, P; Dehnicke, C; Deisz, R A

    2012-10-25

    Acetylcholine has been implicated in higher cortical functions such as learning, memory and cognition, yet the cellular effects of muscarinic acetylcholine receptor (mAChR) activation are poorly understood in the human cortex. Here we investigated the effect of the mAChR agonist carbachol (CCh) and various mAChR antagonists in human cortical slices (from tissue removed during neurosurgical treatment of epilepsy) by intracellular and extracellular recordings. CCh increased neuronal firing, which was antagonised by atropine (non-selective mAChR antagonist) and pirenzepine (M(1)/M(4) mAChRs antagonist) when applied before or after CCh application. AF-DX 116 (M(2)/M(4) mAChRs antagonist) had no effect on CCh-induced increase of firing. CCh also reduced evoked excitatory postsynaptic potentials (EPSP), and the CCh-induced depression of EPSP was fully reversed by atropine. Pirenzepine reversed the depression of CCh on EPSP, but failed to prevent the depression when applied before CCh. AF-DX 116 prevented the CCh-induced depression of evoked EPSP when applied before CCh. CCh also depressed GABAergic transmission and this effect was antagonised by AF-DX 116. Xanomeline (M(1)/M(4) mAChR agonist) increased neuronal firing and decreased EPSP, but had no effect on GABAergic transmission. Reduction (with linopirdine) and enhancement (with retigabine) of the M-current (mediated by K(V)7 channels), increased and decreased neuronal firing, respectively, but had marginal effects on the evoked EPSP. Our results indicate that three pharmacologically distinct mAChRs modulate neuronal firing, glutamatergic and GABAergic transmissions in the human epileptogenic neocortex. The data are discussed towards possible implications of altered mAChR signalling in hyperexcitability and cognitive functions in the human neocortex. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

  12. Epigenetic and Glucocorticoid Receptor-Mediated Regulation of Glutathione Peroxidase 3 in Lung Cancer Cells

    Science.gov (United States)

    An, Byung Chull; Jung, Nak-Kyun; Park, Chun Young; Oh, In-Jae; Choi, Yoo-Duk; Park, Jae-Il; Lee, Seung-won

    2016-01-01

    Glutathione peroxidase 3 (GPx3), an antioxidant enzyme, acts as a modulator of redox signaling, has immunomodulatory function, and catalyzes the detoxification of reactive oxygen species (ROS). GPx3 has been identified as a tumor suppressor in many cancers. Although hyper-methylation of the GPx3 promoter has been shown to down-regulate its expression, other mechanisms by which GPx3 expression is regulated have not been reported. The aim of this study was to further elucidate the mechanisms of GPx3 regulation. GPx3 gene analysis predicted the presence of ten glucocorticoid response elements (GREs) on the GPx3 gene. This result prompted us to investigate whether GPx3 expression is regulated by the glucocorticoid receptor (GR), which is implicated in tumor response to chemotherapy. The corticosteroid dexamethasone (Dex) was used to examine the possible relationship between GR and GPx3 expression. Dex significantly induced GPx3 expression in H1299, H1650, and H1975 cell lines, which exhibit low levels of GPx3 expression under normal conditions. The results of EMSA and ChIP-PCR suggest that GR binds directly to GRE 6 and 7, both of which are located near the GPx3 promoter. Assessment of GPx3 transcription efficiency using a luciferase reporter system showed that blocking formation of the GR-GRE complexes reduced luciferase activity by 7–8-fold. Suppression of GR expression by siRNA transfection also induced down-regulation of GPx3. These data indicate that GPx3 expression can be regulated independently via epigenetic or GR-mediated mechanisms in lung cancer cells, and suggest that GPx3 could potentiate glucocorticoid (GC)-mediated anti-inflammatory signaling in lung cancer cells. PMID:27484907

  13. Bradykinin and adenosine receptors mediate desflurane induced postconditioning in human myocardium: role of reactive oxygen species

    Directory of Open Access Journals (Sweden)

    Gérard Jean-Louis

    2010-07-01

    Full Text Available Abstract Background Desflurane during early reperfusion has been shown to postcondition human myocardium, in vitro. We investigated the role of adenosine and bradykinin receptors, and generation of radical oxygen species in desflurane-induced postconditioning in human myocardium. Methods We recorded isometric contraction of human right atrial trabeculae hanged in an oxygenated Tyrode's solution (34 degrees Celsius, stimulation frequency 1 Hz. After a 30-min hypoxic period, desflurane 6% was administered during the first 5 min of reoxygenation. Desflurane was administered alone or with pretreatment of N-mercaptopropionylglycine, a reactive oxygen species scavenger, 8-(p-Sulfophenyltheophylline, an adenosine receptor antagonist, HOE140, a selective B2 bradykinin receptor antagonist. In separate groups, adenosine and bradykinin were administered during the first minutes of reoxygenation alone or in presence of N-mercaptopropionylglycine. The force of contraction of trabeculae was recorded continuously. Developed force at the end of a 60-min reoxygenation period was compared (mean ± standard deviation between the groups by a variance analysis and post hoc test. Results Desflurane 6% (84 ± 6% of baseline enhanced the recovery of force after 60-min of reoxygenation as compared to control group (51 ± 8% of baseline, P N-mercaptopropionylglycine (54 ± 3% of baseline, 8-(p-Sulfophenyltheophylline (62 ± 9% of baseline, HOE140 (58 ± 6% of baseline abolished desflurane-induced postconditioning. Adenosine (80 ± 9% of baseline and bradykinin (83 ± 4% of baseline induced postconditioning (P vs control, N-mercaptopropionylglycine abolished the beneficial effects of adenosine and bradykinin (54 ± 8 and 58 ± 5% of baseline, respectively. Conclusions In vitro, desflurane-induced postconditioning depends on reactive oxygen species production, activation of adenosine and bradykinin B2 receptors. And, the cardioprotective effect of adenosine and bradykinin

  14. Orexin signaling via the orexin 1 receptor mediates operant responding for food reinforcement.

    Science.gov (United States)

    Sharf, Ruth; Sarhan, Maysa; Brayton, Catherine E; Guarnieri, Douglas J; Taylor, Jane R; DiLeone, Ralph J

    2010-04-15

    Orexin (hypocretin) signaling is implicated in drug addiction and reward, but its role in feeding and food-motivated behavior remains unclear. We investigated orexin's contribution to food-reinforced instrumental responding using an orexin 1 receptor (Ox1r) antagonist, orexin -/- (OKO) and littermate wildtype (WT) mice, and RNAi-mediated knockdown of orexin. C57BL/6J (n = 76) and OKO (n = 39) mice were trained to nose poke for food under a variable ratio schedule of reinforcement. After responding stabilized, a progressive ratio schedule was initiated to evaluate motivation to obtain food reinforcement. Blockade of Ox1r in C57BL/6J mice impaired performance under both the variable ratio and progressive ratio schedules of reinforcement, indicating impaired motivational processes. In contrast, OKO mice initially demonstrated a delay in acquisition but eventually achieved levels of responding similar to those observed in WT animals. Moreover, OKO mice did not differ from WT mice under a progressive ratio schedule, indicating delayed learning processes but no motivational impairments. Considering the differences between pharmacologic blockade of Ox1r and the OKO mice, animals with RNAi mediated knockdown of orexin were then generated and analyzed to eliminate possible developmental effects of missing orexin. Orexin gene knockdown in the lateral hypothalamus in C57BL/6J mice resulted in blunted performance under both the variable ratio and progressive ratio schedules, resembling data obtained following Ox1r antagonism. The behavior seen in OKO mice likely reflects developmental compensation often seen in mutant animals. These data suggest that activation of the Ox1r is a necessary component of food-reinforced responding, motivation, or both in normal mice. Copyright 2010 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  15. Swim stress differentially blocks CRF receptor mediated responses in dorsal raphe nucleus.

    Science.gov (United States)

    Lamy, Christophe M; Beck, Sheryl G

    2010-10-01

    Modulation of the serotonergic (5-HT) neurotransmitter system arising from the dorsal raphe nucleus (DR) is thought to support the behavioral effects of swim stress, i.e., immobility. In vivo pharmacological and anatomical studies suggest that corticotropin-releasing factor (CRF) and γ-aminobutyric acid (GABA) synaptic transmission closely interact to set the response of the DR to swim stress. To investigate the cellular basis of these physiological mechanisms the effects of ovine CRF (oCRF) on GABA(A)-dependent miniature inhibitory postsynaptic currents (mIPSCs) in 5-HT and non-5-HT DR neurons in acute mesencephalic slices obtained from rats either naïve or 24h after a 15 min swim stress session were tested. In this study, the effect of swim stress alone was to decrease the holding current, i.e., hyperpolarize the neuron, and to increase the amplitude and charge of mIPSCs recorded from non-5-HT neurons. Ovine CRF (10 nM) induced an increase in mIPSC frequency in 5-HT neurons recorded from naïve rats, an effect that was suppressed by swim stress. The inward current elicited by oCRF in both 5-HT and non-5-HT neurons was also blocked by swim stress. Ovine CRF increased mIPSCs amplitude and charge in both 5-HT and non-5-HT neurons, but this effect was not modified by swim stress. In concert with our previous findings that swim stress decreased input resistance, action potential threshold and action potential duration and increased glutamatergic synaptic activity the overall primary effect of swim stress is to increase the excitability of 5-HT neurons. These data provide a mechanism at the cellular level for the immobility induced by swim stress and identifies critical components of the raphe circuitry responsible for the altered output of 5-HT neurons induced by swim stress.

  16. A novel gliotic P2 receptor mediating cyclooxygenase-2 induction in rat and human astrocytes.

    Science.gov (United States)

    Brambilla, R; Ceruti, S; Malorni, W; Cattabeni, F; Abbracchio, M P

    2000-07-01

    In astrocytic cultures maintained in vitro, a brief challenge with the ATP analog alpha,beta methyleneATP (alpha,betameATP) results, 3 days later, in marked elongation of astrocytic processes, an event that resembles the astrocytic hypertrophy known to occur in vivo during reactive astrogliosis. alpha,beta meATP-induced effects were observed in primary astrocytes obtained from both rat striatum and cortex (a brain area highly involved in chronic neurodegenerative pathologies), as well as in human astrocytoma cells (ADF cells). Purine-induced gliosis could be reversed by the non-selective P2X/P2Y receptor antagonist pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (PPADS), but not by oxidized ATP (an antagonist of the P2X(7) receptor), in line with previous studies of our laboratory suggesting the involvement of a P2Y receptor subtype. Induction of reactive gliosis was preceded by increased expression of cyclooxygenase-2 (COX-2), an enzyme whose excessive activation has been implicated in both acute and chronic neurodegenerative diseases. The selective COX-2 inhibitor NS-398 prevented both purine-induced astrogliosis and the associated COX-2 induction, suggesting that inhibition of the transcription of the COX-2 gene may also contribute to the anti-inflammatory properties of this agent. Significant blockade of both alpha,beta meATP-mediated reactive gliosis and COX-2 induction was also observed with PPADS. These data suggest that COX-2 mediates P2Y receptor-induced reactive astrogliosis, and that antagonists selective for this receptor subtype may represent a novel class of anti-inflammatory agents of potential interest in acute and chronic neurological disorders characterized by an inflammatory component and reactive gliosis.

  17. Thyrotropin modulates receptor-mediated processing of the atrial natriuretic peptide receptor in cultured thyroid cells

    Energy Technology Data Exchange (ETDEWEB)

    Tseng, Y.L.; Burman, K.D.; Lahiri, S.; Abdelrahim, M.M.; D' Avis, J.C.; Wartofsky, L. (Walter Reed Army Medical Center, Washington, DC (USA))

    1991-03-01

    In a prior study of atrial natriuretic peptide (ANP) binding to cultured thyroid cells, we reported that at 4 C, more than 95% of bound ANP is recovered on cell membranes, with negligible ANP internalization observed. Since ANP binding was inhibited by TSH, we have further studied TSH effects on postbinding ANP processing to determine whether this phenomenon reflects enhanced endocytosis of the ANP-receptor complex. An ANP chase study was initiated by binding (125I) ANP to thyroid cells at 4 C for 2 h, followed by incubation at 37 C. ANP processing was then traced by following 125I activity at various time intervals in three fractions: cell surface membranes, incubation medium, and inside the cells. Radioactivity released into medium represented processed ANP rather than ANP dissociated from surface membranes, since prebound (125I)ANP could not be competitively dissociated by a high concentration of ANP (1 mumol/L) at 37 C. Chase study results showed that prebound ANP quickly disappeared from cell membranes down to 34% by 30 min. Internalized ANP peaked at 10 min, with 21% of initial prebound ANP found inside the cells. At the same time, radioactivity recovered in incubation medium sharply increased between 10-30 min from 8% to 52%. Preincubation of cells with chloroquine (which blocks degradation of the ANP-receptor complex by inhibiting lysosomal hydrolase) caused a 146% increase in internalized (125I)ANP by 30 min (39% compared to 15% control), while medium radioactivity decreased from 52% to 16%, suggesting that processing of the receptor complex is mediated via lysosomal enzymes. In chase studies employing cells pretreated with chloroquine, TSH stimulated the internalization rate of ANP-receptor complex. By 30 min, TSH significantly reduced the membrane-bound ANP, and the decrease was inversely correlated to the increase in internalized radioactivity.

  18. Regulation of AMPA and NMDA receptor-mediated EPSPs in dendritic trees of thalamocortical cells.

    Science.gov (United States)

    Lajeunesse, Francis; Kröger, Helmut; Timofeev, Igor

    2013-01-01

    Two main excitatory synapses are formed at the dendritic arbor of first-order nuclei thalamocortical (TC) neurons. Ascending sensory axons primarily establish contacts at large proximal dendrites, whereas descending corticothalamic fibers form synapses on thin distal dendrites. With the use of a multicomparment computational model based on fully reconstructed TC neurons from the ventroposterolateral nucleus of the cat, we compared local responses at the site of stimulation as well as somatic responses induced by both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)- and N-methyl-D-aspartate receptor (NMDAR)-mediated currents. We found that AMPAR-mediated responses, when synapses were located at proximal dendrites, induced a larger depolarization at the level of soma, whereas NMDAR-mediated responses were more efficient for synapses located at distal dendrites. The voltage transfer and transfer impedance were higher for NMDAR than for AMPAR activation at any location. For both types of synaptic current and for both input locations at the dendritic arbor, somatic responses were characterized by a low variability despite the large variability found in local responses in dendrites. The large neurons had overall smaller somatic responses than small neurons, but this relation was not found in local dendritic responses. We conclude that in TC cells, the dendritic location of small synaptic inputs does not play a major role in the amplitude of a somatic response, but the size of the neuron does. The variability of response amplitude between cells was much larger than the variability within cells. This suggests possible functional segregation of TC neurons of different size.

  19. Purinergic receptor-mediated intracellular Ca2+ oscillations in chicken granulosa cells.

    Science.gov (United States)

    Morley, P; Vanderhyden, B C; Tremblay, R; Mealing, G A; Durkin, J P; Whitfield, J F

    1994-03-01

    production. These studies demonstrate that chicken granulosa cells display P2 purinergic receptors on their surfaces. Activation of these receptors triggers [Ca2+]i oscillations that follow the release of Ca2+ from internal stores and depend on Ca2+ influx through dihydropyridine-insensitive Ca2+ channels. The physiological function(s) of P2 purinergic receptors on granulosa cells is not known.

  20. Receptor-mediated hepatic uptake of M6P-BSA-conjugated triplex-forming oligonucleotides in rats.

    Science.gov (United States)

    Ye, Zhaoyang; Cheng, Kun; Guntaka, Ramareddy V; Mahato, Ram I

    2006-01-01

    Excessive production of extracellular matrix, predominantly type I collagen, results in liver fibrosis. Earlier we synthesized mannose 6-phosphate-bovine serum albumin (M6P-BSA) and conjugated to the type I collagen specific triplex-forming oligonucleotide (TFO) for its enhanced delivery to hepatic stellate cells (HSCs), which is the principal liver fibrogenic cell. In this report, we demonstrate a time-dependent cellular uptake of M6P-BSA-33P-TFO by HSC-T6 cells. Both cellular uptake and nuclear deposition of M6P-BSA-33P-TFO were significantly higher than those of 33P-TFO, leading to enhanced inhibition of type I collagen transcription. Following systemic administration into rats, hepatic accumulation of M6P-BSA-33P-TFO increased from 55% to 68% with the number of M6P per BSA from 14 to 27. Unlike 33P-TFO, there was no significant decrease in the hepatic uptake of (M6P)20-BSA-33P-TFO in fibrotic rats. Prior administration of excess M6P-BSA decreased the hepatic uptake of (M6P)20-BSA-33P-TFO from 66% to 40% in normal rats, and from 60% to 15% in fibrotic rats, suggesting M6P/insulin-like growth factor II (M6P/IGF II) receptor-mediated endocytosis of M6P-BSA-33P-TFO by HSCs. Almost 82% of the total liver uptake in fibrotic rats was contributed by HSCs. In conclusion, by conjugation with M6P-BSA, the TFO could be potentially used for the treatment of liver fibrosis.

  1. Amyloid β-protein differentially affects NMDA receptor- and GABAA receptor-mediated currents in rat hippocampal CA1 neurons

    Institute of Scientific and Technical Information of China (English)

    Junfang Zhang; Lei Hou; Xiuping Gao; Fen Guo; Wei Jing; Jinshun Qi; Jiantian Qiao

    2009-01-01

    Although the aggregated amyloid β-protein (Aβ) in senile plaques is one of the key neuropathological features of Alzheimer's disease (AD), soluble forms of Aβ also interfere with synaptic plasticity at the early stage of AD. The suppressive action of acute application of Aβ on hippocampal long-term potentiation (LTP) has been reported widely, whereas the mechanism underlying the effects of Aβ is still mostly unknown. The present study, using the whole-cell patch clamp technique, investigated the effects of Aβ fragments (Aβ25-35 and Aβ31-35) on the LTP induction-related postsynaptic ligand-gated channel currents in isolated hippocampal CA1 neurons. The results showed a rapid but opposite action of both peptides on excitatory and inhibitory receptor currents. Glutamate application-induced currents were suppressed by A β25-35 in a dose-dependent manner, and further N-methyl-I>aspartate (NMDA) receptor-mediated currents were selec-tively inhibited. In contrast, pretreatment with Aβ fragments potentiated γ-aminobutyric acid (GABA)-induced whole-cell currents. As a control, Aβ35-31 the reversed sequence of Aβ35-31 showed no effect on the currents induced by glutamate, NMDA or GABA. These results may partly explain the impaired effects of Aβ on hippocampal LTP, and suggest that the functional down-regulation of N M DA receptors and up-regulation of GABAA receptors may play an important role in remodeling the hippocampal synaptic plasticity in early AD.

  2. Pentosan polysulfate regulates scavenger receptor-mediated, but not fluid-phase, endocytosis in immortalized cerebral endothelial cells.

    Science.gov (United States)

    Deli, M A; Abrahám, C S; Takahata, H; Katamine, S; Niwa, M

    2000-12-01

    1. Effects of pentosan polysulfate (PPS) and the structurally related sulfated polyanions dextran sulfate, fucoidan, and heparin on the scavenger receptor-mediated and fluidphase endocytosis in GP8 immortalized rat brain endothelial cells were investigated. 2. Using 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarboxyamine perchlorate-labeled acetylated low-density lipoprotein (DiI-AcLDL), we found a binding site with high affinity and low binding capacity, and another one with low affinity and high binding capacity. Increasing ligand concentrations could not saturate DiI-AcLDL uptake. DiI-AcLDL uptake, but not binding, was sensitive to pretreatment with filipin, an inhibitor of caveola formation. 3. PPS (20-200 microg/ml) significantly reduced the binding of DiI-AcLDL after coincubation for 3 hr, though this effect was less expressed after 18 hr. Among other polyanions, only fucoidan decreased the DiI-AcLDL binding after 3 hr, whereas dextran sulfate significantly increased it after 18 hr. PPS treatment induced an increase in DiI-AcLDL uptake, whereas other polysulfated compounds caused a significant reduction. 4. Fluid-phase endocytosis determined by the accumulation of Lucifer yellow was concentration and time dependent in GP8 cells. Coincubation with PPS or other sulfated polyanions could not significantly alter the rate of Lucifer yellow uptake. 5. In conclusion. PPS decreased the binding and increased the uptake of DiI-AcLDL in cerebral endothelial cells, an effect not mimicked by the other polyanions investigated.

  3. ZFAT plays critical roles in peripheral T cell homeostasis and its T cell receptor-mediated response

    Energy Technology Data Exchange (ETDEWEB)

    Doi, Keiko [Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka (Japan); Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka (Japan); Central Research Institute of Life Sciences for the Next Generation of Women Scientists, Fukuoka University, Fukuoka (Japan); Fujimoto, Takahiro [Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka (Japan); Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka (Japan); Okamura, Tadashi [Division of Animal Models, Department of Infectious Diseases, Research Institute, National Center for Global Health and Medicine, Tokyo (Japan); Ogawa, Masahiro [Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka (Japan); Tanaka, Yoko [Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka (Japan); Mototani, Yasumasa; Goto, Motohito [Division of Animal Models, Department of Infectious Diseases, Research Institute, National Center for Global Health and Medicine, Tokyo (Japan); Ota, Takeharu; Matsuzaki, Hiroshi [Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka (Japan); Kuroki, Masahide [Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka (Japan); Tsunoda, Toshiyuki [Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka (Japan); Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka (Japan); Sasazuki, Takehiko [Institute for Advanced Study, Kyushu University, Fukuoka (Japan); Shirasawa, Senji, E-mail: sshirasa@fukuoka-u.ac.jp [Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka (Japan); Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka (Japan)

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer We generated Cd4-Cre-mediated T cell-specific Zfat-deficient mice. Black-Right-Pointing-Pointer Zfat-deficiency leads to reduction in the number of the peripheral T cells. Black-Right-Pointing-Pointer Impaired T cell receptor-mediated response in Zfat-deficient peripheral T cells. Black-Right-Pointing-Pointer Decreased expression of IL-7R{alpha}, IL-2R{alpha} and IL-2 in Zfat-deficient peripheral T cells. Black-Right-Pointing-Pointer Zfat plays critical roles in peripheral T cell homeostasis. -- Abstract: ZFAT, originally identified as a candidate susceptibility gene for autoimmune thyroid disease, has been reported to be involved in apoptosis, development and primitive hematopoiesis. Zfat is highly expressed in T- and B-cells in the lymphoid tissues, however, its physiological function in the immune system remains totally unknown. Here, we generated the T cell-specific Zfat-deficient mice and demonstrated that Zfat-deficiency leads to a remarkable reduction in the number of the peripheral T cells. Intriguingly, a reduced expression of IL-7R{alpha} and the impaired responsiveness to IL-7 for the survival were observed in the Zfat-deficient T cells. Furthermore, a severe defect in proliferation and increased apoptosis in the Zfat-deficient T cells following T cell receptor (TCR) stimulation was observed with a reduced IL-2R{alpha} expression as well as a reduced IL-2 production. Thus, our findings reveal that Zfat is a critical regulator in peripheral T cell homeostasis and its TCR-mediated response.

  4. Prostaglandin E2 induces vascular relaxation by E-prostanoid 4 receptor-mediated activation of endothelial nitric oxide synthase

    DEFF Research Database (Denmark)

    Hristovska, Ana-Marija; Rasmussen, Lasse E; Hansen, Pernille B L;

    2007-01-01

    , calyculin (10(-8) mol/L), abolished the PGE(2)-mediated relaxation. In aortic rings, PGE(2) dephosphorylated eNOS at Thr(495). Chronically catheterized eNOS(-/-) mice were hypertensive (137+/-3.6 mm Hg, n=13, versus 101+/-3.9 mm Hg, n=9) and exhibited a lower sensitivity of blood pressure reduction...

  5. Development of drug loaded nanoparticles for tumor targeting. Part 2: Enhancement of tumor penetration through receptor mediated transcytosis in 3D tumor models

    Science.gov (United States)

    El-Dakdouki, Mohammad H.; Puré, Ellen; Huang, Xuefei

    2013-04-01

    We report that receptor mediated transcytosis can be utilized to facilitate tumor penetration by drug loaded nanoparticles (NPs). We synthesized hyaluronan (HA) coated silica nanoparticles (SNPs) containing a highly fluorescent core to target CD44 expressed on the cancer cell surface. Although prior studies have primarily focused on CD44 mediated endocytosis to facilitate cellular uptake of HA-NPs by cancer cells, we discovered that, once internalized, the HA-SNPs could be transported out of the cells with their cargo. The exported NPs could be taken up by neighboring cells. This enabled the HA-SNPs to penetrate deeper inside tumors and reach a much greater number of tumor cells in 3D tumor models, presumably through tandem cycles of CD44 mediated endocytosis and exocytosis. When doxorubicin (DOX) was loaded onto the NPs, better penetration of multilayered tumor cells was observed with much improved cytotoxicities against both drug sensitive and drug resistant cancer spheroids compared to the free drug. Thus, targeting receptors such as CD44 that can readily undergo recycling between the cell surface and interior of the cells can become a useful strategy to enhance the tumor penetration potential of NPs and the efficiency of drug delivery through receptor mediated transcytosis.We report that receptor mediated transcytosis can be utilized to facilitate tumor penetration by drug loaded nanoparticles (NPs). We synthesized hyaluronan (HA) coated silica nanoparticles (SNPs) containing a highly fluorescent core to target CD44 expressed on the cancer cell surface. Although prior studies have primarily focused on CD44 mediated endocytosis to facilitate cellular uptake of HA-NPs by cancer cells, we discovered that, once internalized, the HA-SNPs could be transported out of the cells with their cargo. The exported NPs could be taken up by neighboring cells. This enabled the HA-SNPs to penetrate deeper inside tumors and reach a much greater number of tumor cells in 3D tumor

  6. Antagonism of GABAB-receptor-mediated responses in the guinea-pig isolated ileum and vas deferens by phosphono-analogues of GABA.

    OpenAIRE

    Kerr, D. I.; Ong, J; Prager, R. H.

    1990-01-01

    1. The phosphono-analogues of gamma-aminobutyric acid (GABA), 4-amino-butylphosphonic acid (4-ABPA), 3-amino-2-(4-chlorophenyl)-propylphosphonic acid (phaclofen) and 3-amino-2-cyclohexylpropyl-phosphonic acid, each antagonized the GABA- and baclofen-induced GABAB-receptor-mediated depression of twitch responses to transmural stimulation in the guinea-pig isolated ileum, in a concentration-dependent, reversible and surmountable manner (apparent pA2 = 4.0 +/- 0.1, 4 +/- 0.2 and 3.7 +/- 0.2 resp...

  7. Quantitation of the Contractile Response Mediated by Two Receptors: M2 and M3 Muscarinic Receptor-Mediated Contractions of Human Gastroesophageal Smooth MuscleS⃞

    Science.gov (United States)

    Braverman, Alan S.; Miller, Larry S.; Vegesna, Anil K.; Tiwana, Mansoor I.; Tallarida, Ronald J.; Ruggieri, Michael R.

    2009-01-01

    Although muscarinic receptors are known to mediate tonic contraction of human gastrointestinal tract smooth muscle, the receptor subtypes that mediate the tonic contractions are not entirely clear. Whole human stomachs with attached esophagus were procured from organ transplant donors. Cholinergic contractile responses of clasp, sling, lower esophageal circular (LEC), midesophageal circular (MEC), and midesophageal longitudinal (MEL) muscle strips were determined. Sling fibers contracted greater than the other fibers. Total, M2 and M3 muscarinic receptor density was determined for each of these dissections by immunoprecipitation. M2 receptor density is greatest in the sling fibers, followed by clasp, LEC, MEC, and then MEL, whereas M3 density is greatest in LEC, followed by MEL, MEC, sling, and then clasp. The potency of subtype-selective antagonists to inhibit bethanechol-induced contraction was calculated by Schild analysis to determine which muscarinic receptor subtypes contribute to contraction. The results suggest both M2 and M3 receptors mediate contraction in clasp and sling fibers. Thus, this type of analysis in which multiple receptors mediate the contractile response is inappropriate, and an analysis method relating dual occupation of M2 and M3 receptors to contraction is presented. Using this new method of analysis, it was found that the M2 muscarinic receptor plays a greater role in mediating contraction of clasp and sling fibers than in LEC, MEC, and MEL muscles in which the M3 receptor predominantly mediates contraction. PMID:19126780

  8. Quantitation of the contractile response mediated by two receptors: M2 and M3 muscarinic receptor-mediated contractions of human gastroesophageal smooth muscle.

    Science.gov (United States)

    Braverman, Alan S; Miller, Larry S; Vegesna, Anil K; Tiwana, Mansoor I; Tallarida, Ronald J; Ruggieri, Michael R

    2009-04-01

    Although muscarinic receptors are known to mediate tonic contraction of human gastrointestinal tract smooth muscle, the receptor subtypes that mediate the tonic contractions are not entirely clear. Whole human stomachs with attached esophagus were procured from organ transplant donors. Cholinergic contractile responses of clasp, sling, lower esophageal circular (LEC), midesophageal circular (MEC), and midesophageal longitudinal (MEL) muscle strips were determined. Sling fibers contracted greater than the other fibers. Total, M(2) and M(3) muscarinic receptor density was determined for each of these dissections by immunoprecipitation. M(2) receptor density is greatest in the sling fibers, followed by clasp, LEC, MEC, and then MEL, whereas M(3) density is greatest in LEC, followed by MEL, MEC, sling, and then clasp. The potency of subtype-selective antagonists to inhibit bethanechol-induced contraction was calculated by Schild analysis to determine which muscarinic receptor subtypes contribute to contraction. The results suggest both M(2) and M(3) receptors mediate contraction in clasp and sling fibers. Thus, this type of analysis in which multiple receptors mediate the contractile response is inappropriate, and an analysis method relating dual occupation of M(2) and M(3) receptors to contraction is presented. Using this new method of analysis, it was found that the M(2) muscarinic receptor plays a greater role in mediating contraction of clasp and sling fibers than in LEC, MEC, and MEL muscles in which the M(3) receptor predominantly mediates contraction.

  9. Changes in synaptic and extrasynaptic N-methyl-D-aspartate receptor-mediated currents at early-stage epileptogenesis in adult mice

    Institute of Scientific and Technical Information of China (English)

    Juegang Ju; Sheng-tian Li

    2011-01-01

    Previous reports have shown that N-methyl-D-aspartate (NMDA) receptors are extensively involved in epilepsy genesis and recurrence.Recent studies have shown that synaptic and extrasynaptic NMDA receptors play different, or even opposing, roles in various signaling pathways, including synaptic plasticity and neuronal death.The present study analyzed changes in synaptic and extrasynaptic NMDA receptor-mediated currents during epilepsy onset.Mouse models of lithium chloride pilocarpine-induced epilepsy were established, and hippocampal slices were prepared at 24 hours after the onset of status epilepticus.Synaptic and extrasynaptic NMDA receptor-mediated excitatory post-synaptic currents (NMDA-EPSCs) were recorded in CA1 pyramidal neurons by whole-cell patch clamp technique.Results demonstrated no significant difference in rise and delay time of synaptic NMDA-EPSCs compared with normal neurons.Peak amplitude, area-to-peak ratio,and rising time of extrasynaptic NMDA-EPSCs remained unchanged, but decay of extrasynaptic NMDA-EPSCs was faster than that of normal neurons.These results suggest that extrasynaptic NMDA receptors play a role in epileptogenesis.

  10. Nicotine effects on muscarinic receptor-mediated free Ca[Formula: see text] level changes in the facial nucleus following facial nerve injury.

    Science.gov (United States)

    Sun, Dawei; Zhou, Rui; Dong, Anbing; Sun, Wenhai; Zhang, Hongmei; Tang, Limin

    2016-06-01

    It was suggested that muscarinic, and nicotinic receptors increase free Ca[Formula: see text] levels in the facial nerve nucleus via various channels following facial nerve injury. However, intracellular Ca[Formula: see text] overload can trigger either necrotic or apoptotic cell death. It is assumed that, following facial nerve injury, the interactions of nicotinic and muscarinic acetylcholine receptors in facial nerve nucleus may negatively regulate free Ca[Formula: see text] concentrations in the facial nerve nucleus, which provide important information for the repair and regeneration of the facial nerve. The present study investigated the regulatory effects of nicotine on muscarinic receptor-mediated free calcium ion level changes in the facial nucleus in a rat model of facial nerve injury at 7, 30, and 90 days following facial nerve injury using laser confocal microscopy. The dose-dependent regulation of nicotine on muscarinic receptor-mediated free calcium ion level changes in the facial nucleus may decrease the range of free Ca[Formula: see text] increases following facial nerve injury, which is important for nerve cell regeneration. It is concluded that the negative effects of nicotine on muscarinic receptors are related to the [Formula: see text] subtype of nicotinic receptors.

  11. Blocking GABA(A) inhibition reveals AMPA- and NMDA-receptor-mediated polysynaptic responses in the CA1 region of the rat hippocampus.

    Science.gov (United States)

    Crépel, V; Khazipov, R; Ben-Ari, Y

    1997-04-01

    We have investigated the conditions required to evoke polysynaptic responses in the isolated CA1 region of hippocampal slices from Wistar adult rats. Experiments were performed with extracellular and whole cell recording techniques. In the presence of bicuculline (10 microM), 6-cyano-7-nitroquinoxaline-2-3-dione (10 microM), glycine (10 microM), and a low external concentration of Mg2+ (0.3 mM), electrical stimulation of the Schaffer collaterals/commissural pathway evoked graded N-methyl-D-aspartate (NMDA)-receptor-mediated late field potentials in the stratum radiatum of the CA1 region. These responses were generated via polysynaptic connections because their latency varied strongly and inversely with the stimulation intensity and they were abolished by a high concentration of divalent cations (7 mM Ca2+). These responses likely were driven by local collateral branches of CA1 pyramidal cell axons because focal application of tetrodotoxin (30 microM) in the stratum oriens strongly reduced the late synaptic component and antidromic stimulation of CA1 pyramidal cells could evoke the polysynaptic response. Current-source density analysis suggested that the polysynaptic response was generated along the proximal part of the apical dendrites of CA1 pyramidal cells (50-150 microm below the pyramidal cell layer in the stratum radiatum). In physiological concentration of Mg2+ (1.3 mM), the pharmacologically isolated NMDA-receptor-mediated polysynaptic response was abolished. In control artificial cerebrospinal fluid (with physiological concentration of Mg2+), bicuculline ( 10 microM) generated a graded polysynaptic response. Under these conditions, this response was mediated both by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/NMDA receptors. In the presence of D-2-amino-5-phosphonovalerate (50 microM), the polysynaptic response could be mediated by AMPA receptors, although less efficiently. In conclusion, suppression of gamma-aminobutyric acid

  12. Long-term exposure to IL-1beta enhances Toll-IL-1 receptor-mediated inflammatory signaling in murine airway hyperresponsiveness

    DEFF Research Database (Denmark)

    Zhang, Yaping; Xu, Cang-Bao; Cardell, Lars-Olaf

    2009-01-01

    Toll-interleukin-1 (Toll-IL-1) receptor signaling may play a key role in the development of airway hyperreactivity (AHR) and chronic airway inflammatory diseases such as asthma. Previously, we have demonstrated that pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin......-time PCR-based cDNA array. The key gene expressions that were altered were verified by immunohistochemistry using confocal microscopy. Tracheal ring segment contractile responsiveness to the inflammatory mediator bradykinin was monitored using a sensitive myograph system. The results showed that after......-1beta (IL-1beta), induce AHR. However, the underlying intracellular signaling mechanisms that lead to AHR remain elusive. In order to see if the Toll-IL-1 receptor-mediated inflammatory signal pathways are involved in the development of AHR, the present study was designed to use a real-time PCR...

  13. Effects of gamma-aminobutyric acid receptors on muscarinic receptor-mediated free calcium ion levels in the facial nucleus following facial nerve injury

    Institute of Scientific and Technical Information of China (English)

    Guangfeng Jiang; Dawei Sun; Rui Zhou; Fugao Zhu; Yanqing Wang; Xiuming Wan; Banghua Liu

    2011-01-01

    Muscarinic receptors and nicotine receptors can increase free calcium ion levels in the facial nucleus via different channels following facial nerve injury. In addition, γ-aminobutyric acid A (GABAA) receptors have been shown to negatively regulate free calcium ion levels in the facial nucleus by inhibiting nicotine receptors. The present study investigated the influence of GABAA, γ-aminobutyric acid B (GABAB) and C (GABAC) receptors on muscarinic receptors in rats with facial nerve injury by confocal laser microscopy. GABAA and GABAB receptors exhibited significant dose-dependent inhibitory effects on increased muscarinic receptor-mediated free calcium ion levels following facial nerve injury. Results showed that GABAA and GABAB receptors negatively regulate muscarinic receptor effects and interplay with cholinergic receptors to regulate free calcium ion levels for facial neural regeneration.

  14. GABAA receptor-mediated feedforward and feedback inhibition differentially modulate the gain and the neural code transformation in hippocampal CA1 pyramidal cells.

    Science.gov (United States)

    Jang, Hyun Jae; Park, Kyerl; Lee, Jaedong; Kim, Hyuncheol; Han, Kyu Hun; Kwag, Jeehyun

    2015-12-01

    Diverse variety of hippocampal interneurons exists in the CA1 area, which provides either feedforward (FF) or feedback (FB) inhibition to CA1 pyramidal cell (PC). However, how the two different inhibitory network architectures modulate the computational mode of CA1 PC is unknown. By investigating the CA3 PC rate-driven input-output function of CA1 PC using in vitro electrophysiology, in vitro-simulation of inhibitory network, and in silico computational modeling, we demonstrated for the first time that GABAA receptor-mediated FF and FB inhibition differentially modulate the gain, the spike precision, the neural code transformation and the information capacity of CA1 PC. Recruitment of FF inhibition buffered the CA1 PC spikes to theta-frequency regardless of the input frequency, abolishing the gain and making CA1 PC insensitive to its inputs. Instead, temporal variability of the CA1 PC spikes was increased, promoting the rate-to-temporal code transformation to enhance the information capacity of CA1 PC. In contrast, the recruitment of FB inhibition sub-linearly transformed the input rate to spike output rate with high gain and low spike temporal variability, promoting the rate-to-rate code transformation. These results suggest that GABAA receptor-mediated FF and FB inhibitory circuits could serve as network mechanisms for differentially modulating the gain of CA1 PC, allowing CA1 PC to switch between different computational modes using rate and temporal codes ad hoc. Such switch will allow CA1 PC to efficiently respond to spatio-temporally dynamic inputs and expand its computational capacity during different behavioral and neuromodulatory states in vivo.

  15. 5-HT7 receptor-mediated meningeal dilatation induced by 5-carboxamidotryptamine in rats is not altered by 5-HT depletion and chronic corticosterone treatment.

    Science.gov (United States)

    Martínez-García, E; Sánchez-Maldonado, C; Terrón, J A

    2011-01-01

    Low brain serotonin levels and high circulating levels of corticosterone are features of migraine. The 5-HT7 receptor was shown to mediate dilator responses to the 5-HT1B/1D and 5-HT7 receptor agonist, 5-carboxamidotryptamine in the middle meningeal artery of rats. Here we analyzed the effect of serotonin depletion and chronic corticosterone treatment on 5-HT7 receptor-mediated dilatation induced by 5-carboxamidotryptamine in the middle meningeal artery of anesthetized rats. Two weeks before experiments, male Wistar rats received i.c.v. injections of vehicle or the neurotoxin, 5,7-dihydroxytryptamine; upon recovery, animals received a chronic s.c. treatment (2 weeks) with vehicle (1 ml/kg/day) or corticosterone (20 mg/kg/day). At the end of treatments, animals were anesthetized and prepared for recording of blood pressure and blood flow in the middle meningeal artery, and i.v. drug administration. All animals received the 5-HT1B/1D receptor antagonist GR-127935 (1 mg/kg, i.v.) alone or combined with the 5-HT7 receptor antagonist, SB-269970 (1 mg/kg, i.v.). Topical 5-carboxamidotryptamine (0.01-1000 microM) to the exposed dura mater encephala produced decreases in diastolic blood pressure, variable changes in meningeal blood flow and increases in conductance (i.e. dilatation) in the middle meningeal artery. Meningeal dilator responses to 5-carboxamidotryptamine did not differ among treatment groups. In all cases, the combined treatment with GR-127935 + SB-269970 inhibited hypotensive and meningeal dilator responses to 5- carboxamidotryptamine. Together, these data do not support the notion that 5-HT7 receptors mediating dilatation in the middle meningeal artery are regulated by low brain serotonin levels and/or chronically high circulating levels of corticosterone. Further studies are required to elucidate the potential impact of these conditions and the role of 5-HT7 receptors in migraine.

  16. Prenatal nicotine is associated with reduced AMPA and NMDA receptor-mediated rises in calcium within the laterodorsal tegmentum: a pontine nucleus involved in addiction processes.

    Science.gov (United States)

    McNair, L F; Kohlmeier, K A

    2015-06-01

    Despite huge efforts from public sectors to educate society as to the deleterious physiological consequences of smoking while pregnant, 12-25% of all babies worldwide are born to mothers who smoked during their pregnancies. Chief among the negative legacies bestowed to the exposed individual is an enhanced proclivity postnatally to addict to drugs of abuse, which suggests that the drug exposure during gestation changed the developing brain in such a way that biased it towards addiction. Glutamate signalling has been shown to be altered by prenatal nicotine exposure (PNE) and glutamate is the major excitatory neurotransmitter within the laterodorsal tegmental nucleus (LDT), which is a brainstem region importantly involved in responding to motivational stimuli and critical in development of drug addiction-associated behaviours, however, it is unknown whether PNE alters glutamate signalling within this nucleus. Accordingly, we used calcium imaging, to evaluate AMPA and NMDA receptor-mediated calcium responses in LDT brain slices from control and PNE mice. We also investigated whether the positive AMPA receptor modulator cyclothiazide (CYZ) had differential actions on calcium in the LDT following PNE. Our data indicated that PNE significantly decreased AMPA receptor-mediated calcium responses, and altered the neuronal calcium response to consecutive NMDA applications within the LDT. Furthermore, CYZ strongly potentiated AMPA-induced responses, however, this action was significantly reduced in the LDT of PNE mice when compared with enhancements in responses in control LDT cells. Immunohistochemical processing confirmed that calcium imaging recordings were obtained from the LDT nucleus as determined by presence of cholinergic neurons. Our results contribute to the body of evidence suggesting that neurobiological changes are induced if gestation is accompanied by nicotine exposure. We conclude that in light of the role played by the LDT in motivated behaviour, the

  17. The plasma concentration of HDL-associated apoM is influenced by LDL receptor-mediated clearance of apoB-containing particles.

    Science.gov (United States)

    Christoffersen, Christina; Benn, Marianne; Christensen, Pernille M; Gordts, Philip L S M; Roebroek, Anton J M; Frikke-Schmidt, Ruth; Tybjaerg-Hansen, Anne; Dahlbäck, Björn; Nielsen, Lars B

    2012-10-01

    ApoM is mainly associated with HDL. Nevertheless, we have consistently observed positive correlations of apoM with plasma LDL cholesterol in humans. Moreover, LDL receptor deficiency is associated with increased plasma apoM in mice. Here, we tested the idea that plasma apoM concentrations are affected by the rate of LDL receptor-mediated clearance of apoB-containing particles. We measured apoM in humans each carrying one of three different LDL receptor mutations (n = 9) or the apoB3500 mutation (n = 12). These carriers had increased plasma apoM (1.34 ± 0.13 µM, P = 0.003, and 1.23 ± 0.10 µM, P = 0.02, respectively) as compared with noncarriers (0.93 ± 0.04 µM). When we injected human apoM-containing HDL into Wt (n = 6) or LDL receptor-deficient mice (n = 6), the removal of HDL-associated human apoM was delayed in the LDL receptor-deficient mice. After 2 h, 54 ± 5% versus 90 ± 8% (P LDL receptor-deficient mice, respectively. Finally, we compared the turnover of radio-iodinated LDL and plasma apoM concentrations in 45 normocholesterolemic humans. There was a negative correlation between plasma apoM and the fractional catabolic rate of LDL (r = -0.38, P = 0.009). These data suggest that the plasma clearance of apoM, despite apoM primarily being associated with HDL, is influenced by LDL receptor-mediated clearance of apoB-containing particles.

  18. Chemical kinetic measurements of the effect of trans- and cis-3,3'-Bis[(trimethylammonio)methyl]azobenzene bromide on acetylcholine receptor mediated ion translocation in Electrophorus electricus and Torpedo californica.

    Science.gov (United States)

    Delcour, A H; Hess, G P

    1986-04-08

    A quench-flow technique was used to study the effect of trans- and cis-3,3'-bis[(trimethylammonio)methyl]azobenzene bromide (trans- and cis-Bis-Q), photoisomerizable ligands, on the acetylcholine receptor in vesicles prepared from the electric organ of Electrophorus electricus and of Torpedo californica. In E. electricus, two rate coefficients of the receptor-mediated translocation of 86Rb+ induced with trans-Bis-Q were measured: JA, the rate coefficient for ion flux, and alpha, the rate coefficient for receptor inactivation (desensitization). Both rate coefficients increase with increasing concentrations of Bis-Q up to 50 microM. At higher concentrations JA decreases in a concentration-dependent manner while alpha remains unchanged. This effect was previously observed with suberyldicholine [Pasquale, E. B., Takeyasu, K., Udgaonkar, J., Cash, D.J., Severski, M.C., & Hess, G. P. (1983) Biochemistry 22, 5967-5973] and with acetylcholine [Takeyasu, K., Udgaonkar, J., & Hess, G. P. (1983) Biochemistry 22, 5973-5978] and was analyzed in terms of a minimum mechanism that accounts for the properties of activation, desensitization, and inhibition of the receptor. Two molecules of trans-Bis-Q must be bound for the channel to open, but at concentrations greater than 50 microM the population of open channels decreases because of the additional binding of one molecule of trans-Bis-Q to a regulatory inhibitory site, independent of the activating sites. cis-Bis-Q does not induce transmembrane ion flux, but it does inhibit the response of the receptor to acetylcholine and induces inactivation (desensitization) in the micromolar range.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Serotonin 5-HT2C receptor-mediated inhibition of the M-current in hypothalamic POMC neurons

    OpenAIRE

    Roepke, T. A.; Smith, A W; Rønnekleiv, O. K.; Kelly, M. J.

    2012-01-01

    Hypothalamic proopiomelanocortin (POMC) neurons are controlled by many central signals, including serotonin. Serotonin increases POMC activity and reduces feeding behavior via serotonion [5-hydroxytryptamine (5-HT)] receptors by modulating K+ currents. A potential K+ current is the M-current, a noninactivating, subthreshold outward K+ current. Previously, we found that M-current activity was highly reduced in fasted vs. fed states in neuropeptide Y neurons. Because POMC neurons also respond t...

  20. Control of Spike Transfer at Hippocampal Mossy Fiber Synapses In Vivo by GABAA and GABAB Receptor-Mediated Inhibition.

    Science.gov (United States)

    Zucca, Stefano; Griguoli, Marilena; Malézieux, Meryl; Grosjean, Noëlle; Carta, Mario; Mulle, Christophe

    2017-01-18

    Despite extensive studies in hippocampal slices and incentive from computational theories, the synaptic mechanisms underlying information transfer at mossy fiber (mf) connections between the dentate gyrus (DG) and CA3 neurons in vivo are still elusive. Here we used an optogenetic approach in mice to selectively target and control the activity of DG granule cells (GCs) while performing whole-cell and juxtacellular recordings of CA3 neurons in vivo In CA3 pyramidal cells (PCs), mf-CA3 synaptic responses consisted predominantly of an IPSP at low stimulation frequency (0.05 Hz). Upon increasing the frequency of stimulation, a biphasic response was observed consisting of a brief mf EPSP followed by an inhibitory response lasting on the order of 100 ms. Spike transfer at DG-CA3 interneurons recorded in the juxtacellular mode was efficient at low presynaptic stimulation frequency and appeared insensitive to an increased frequency of GC activity. Overall, this resulted in a robust and slow feedforward inhibition of spike transfer at mf-CA3 pyramidal cell synapses. Short-term plasticity of EPSPs with increasing frequency of presynaptic activity allowed inhibition to be overcome to reach spike discharge in CA3 PCs. Whereas the activation of GABAA receptors was responsible for the direct inhibition of light-evoked spike responses, the slow inhibition of spiking activity required the activation of GABAB receptors in CA3 PCs. The slow inhibitory response defined an optimum frequency of presynaptic activity for spike transfer at ∼10 Hz. Altogether these properties define the temporal rules for efficient information transfer at DG-CA3 synaptic connections in the intact circuit.

  1. A role for Toll-like receptor mediated signals in neutrophils in the pathogenesis of the anti-phospholipid syndrome.

    Directory of Open Access Journals (Sweden)

    Gerd Gladigau

    Full Text Available The anti-phospholipid syndrome (APS is characterized by recurrent thrombosis and occurrence of anti-phospholipid antibodies (aPL. aPL are necessary, but not sufficient for the clinical manifestations of APS. Growing evidence suggests a role of innate immune cells, in particular polymorphonuclear neutrophils (PMN and Toll-like receptors (TLR to be additionally involved. aPL activate endothelial cells and monocytes through a TLR4-dependent signalling pathway. Whether this is also relevant for PMN in a similar way is currently not known. To address this issue, we used purified PMN from healthy donors and stimulated them in the presence or absence of human monoclonal aPL and the TLR4 agonist LPS monitoring neutrophil effector functions, namely the oxidative burst, phagocytosis, L-Selectin shedding and IL-8 production. aPL alone were only able to induce minor activation of PMN effector functions at high concentrations. However, in the additional presence of LPS the activation threshold was markedly lower indicating a synergistic activation pathway of aPL and TLR in PMN. In summary, our results indicate that PMN effector functions are directly activated by aPL and boosted by the additional presence of microbial products. This highlights a role for PMN as important innate immune effector cells that contribute to the pathophysiology of APS.

  2. (Pro)renin receptor mediates both angiotensin II-dependent and -independent oxidative stress in neuronal cells.

    Science.gov (United States)

    Peng, Hua; Li, Wencheng; Seth, Dale M; Nair, Anand R; Francis, Joseph; Feng, Yumei

    2013-01-01

    The binding of renin or prorenin to the (pro)renin receptor (PRR) promotes angiotensin (Ang) II formation and mediates Ang II-independent signaling pathways. In the central nervous system (CNS), Ang II regulates blood pressure via inducing oxidative stress; however, the role of PRR-mediated Ang II-independent signaling pathways in oxidative stress in the CNS remains undefined. To address this question, Neuro-2A cells were infected with control virus or an adeno-associated virus encoding the human PRR. Human PRR over-expression alone increased ROS levels, NADPH oxidase activity, as well as NADPH oxidase (NOX) isoforms 2 and 4 mRNA expression levels and these effects were not blocked by losartan. Moreover, the increase in NOX 2 and NOX 4 mRNA levels, NADPH oxidase activity, and ROS levels induced by PRR over-expression was prevented by mitogen activated protein kinase/extracellular signal-regulated kinase 1 and 2 (MAPK/ERK1/2) inhibition, and phosphoinositide 3 kinase/Akt (IP3/Akt) inhibition, indicating that PRR regulates NOX activity and ROS formation in neuro-2A cells through Ang II-independent ERK1/2 and IP3/Akt activation. Interestingly, at a concentration of 2 nM or higher, prorenin promoted Ang II formation, and thus further increased the ROS levels in cultured Neuro-2A cells via PRR. In conclusion, human PRR over-expression induced ROS production through both angiotensin II-dependent and -independent mechanisms. We showed that PRR-mediated angiotensin II-independent ROS formation is associated with activation of the MAPK/ERK1/2 and PI3/Akt signaling pathways and up-regulation of mRNA level of NOX 2 and NOX4 isoforms in neuronal cells.

  3. Vitamin D Receptor-Mediated Upregulation of CYP3A4 and MDR1 by Quercetin in Caco-2 cells.

    Science.gov (United States)

    Chae, Yoon-Jee; Cho, Kwan Hyung; Yoon, In-Soo; Noh, Chi-Kyoung; Lee, Hyo-Jong; Park, Yohan; Ji, Eunhee; Seo, Min-Duk; Maeng, Han-Joo

    2016-01-01

    To examine whether quercetin interacts with vitamin D receptor, we investigated the effects of quercetin on vitamin D receptor activity in human intestinal Caco-2 cells. The effects of quercetin on the expression of the vitamin D receptor target genes, vitamin D3 24-hydroxylase, cytochrome P450 3A4, multidrug resistance protein 1, and transient receptor potential vanilloid type 6 were measured using quantitative polymerase chain reaction. The vitamin D receptor siRNA was used to assess the involvement of the vitamin D receptor. Vitamin D receptor activation using a vitamin D responsive element-mediated cytochrome P450 3A4 reporter gene assay was investigated in Caco-2 cells transfected with human vitamin D receptor. We also studied the magnitude of the vitamin D receptor activation and/or synergism between 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] and quercetin-like flavonoids. Slight but significant increases in the mRNA expression of cytochrome P450 3A4, vitamin D3 24-hydroxylase, multidrug resistance protein 1, and transient receptor potential vanilloid type 6 were observed after 3 days of continual quercetin treatment. The silencing effect of vitamin D receptor by vitamin D receptor siRNA in Caco-2 cells significantly attenuated the induction of the vitamin D receptor target genes. Moreover, quercetin significantly enhanced cytochrome P450 3A4 reporter activity in Caco-2 cells in a dose-dependent manner, and the expression of exogenous vitamin D receptor further stimulated the vitamin D receptor activity. Quercetin-like flavonoids such as kaempferol stimulated the vitamin D receptor activity in a manner similar to that seen with quercetin. Taken together, the data indicates that quercetin upregulates cytochrome P450 3A4 and multidrug resistance protein 1 expression in Caco-2 cells likely via a vitamin D receptor-dependent pathway.

  4. Rat NPFF(1) receptor-mediated signaling: functional comparison of neuropeptide FF (NPFF), FMRFamide and PFR(Tic)amide.

    Science.gov (United States)

    Chen, Jin-Chung; Lee, Wei-Hsin; Chen, Pei-Chun; Tseng, Ching-Ping; Huang, Eagle Yi-Kung

    2006-05-01

    Neuropeptide FF (NPFF) participates in many physiological functions associated with opioids in the mammalian CNS. We established a pheochromocytoma PC-12 cell line clone stably expressing rat NPFF1 receptors. Both NPFF and FMRFamide activated NPFF1 receptors to couple with Gi/o protein and inhibited adenylyl cyclase activity in PC-12/rNPFF1 cells, but there were no effects on MAPKs (ERK1/2 and p38 MAPK) or PI3K/Akt pathway. FMRFamide also inhibited DARPP-32/Thr34 phosphorylation in the presence of forskolin. Similarly, PFR(Tic)amide, a 'super-agonist' of NPFF receptors, inhibited the production of cAMP and slightly decreased DARPP-32/Thr34 phosphorylation in PC-12/rNPFF1 cells. Intracerebroventricular injections of PFR(Tic)amide blocked behavioral sensitization of locomotor activity to amphetamine, and intrathecal injection of PFR(Tic)amide caused a dose-dependent antinociception in vivo in rats. Thus, "over-activation" of NPFF receptors by PFR(Tic)amide induced different bio-effects from those induced by NPFF in vivo.

  5. Specific in vitro toxicity of crude and refined petroleum products. 1. Aryl hydrocarbon receptor-mediated responses

    NARCIS (Netherlands)

    Vrabie, C.M.; Jonker, M.T.O.; Murk, A.J.

    2009-01-01

    The present study is the first in a series reporting on in vitro toxic potencies of oils. The objective was to determine whether 11 crude oils and refined products activate the aryl hydrocarbon receptor (AhR) in a dioxin receptor¿mediated luciferase assay. Cells were exposed for 6 and 24 h to differ

  6. Specific in vitro toxicity of crude and refined petroleum products. 1. Aryl hydrocarbon receptor-mediated responses.

    NARCIS (Netherlands)

    Vrabie, C.M.; Jonker, M.T.O.; Murk, A.J.

    2009-01-01

    The present study is the first in a series reporting on in vitro toxic potencies of oils. The objective was to determine whether 11 crude oils and refined products activate the aryl hydrocarbon receptor (AhR) in a dioxin receptor–mediated luciferase assay. Cells were exposed for 6 and 24 h to differ

  7. Specific in vitro toxicity of crude and refined petroleum products. 1. Aryl hydrocarbon receptor-mediated responses

    NARCIS (Netherlands)

    Vrabie, C.M.; Jonker, M.T.O.; Murk, A.J.

    2009-01-01

    The present study is the first in a series reporting on in vitro toxic potencies of oils. The objective was to determine whether 11 crude oils and refined products activate the aryl hydrocarbon receptor (AhR) in a dioxin receptor¿mediated luciferase assay. Cells were exposed for 6 and 24 h to

  8. Major and minor group rhinoviruses elicit differential signaling and cytokine responses as a function of receptor-mediated signal transduction.

    Directory of Open Access Journals (Sweden)

    Bryce A Schuler

    Full Text Available Major- and minor-group human rhinoviruses (HRV enter their host by binding to the cell surface molecules ICAM-1 and LDL-R, respectively, which are present on both macrophages and epithelial cells. Although epithelial cells are the primary site of productive HRV infection, previous studies have implicated macrophages in establishing the cytokine dysregulation that occurs during rhinovirus-induced asthma exacerbations. Analysis of the transcriptome of primary human macrophages exposed to major- and minor-group HRV demonstrated differential gene expression. Alterations in gene expression were traced to differential mitochondrial activity and signaling pathway activation between two rhinovirus serotypes, HRV16 (major-group and HRV1A (minor-group, upon initial HRV binding. Variances in phosphorylation of kinases (p38, JNK, ERK5 and transcription factors (ATF-2, CREB, CEBP-alpha were observed between the major- and minor-group HRV treatments. Differential activation of signaling pathways led to changes in the production of the asthma-relevant cytokines CCL20, CCL2, and IL-10. This is the first report of genetically similar viruses eliciting dissimilar cytokine release, transcription factor phosphorylation, and MAPK activation from macrophages, suggesting that receptor use is a mechanism for establishing the inflammatory microenvironment in the human airway upon exposure to rhinovirus.

  9. Neutrophil P2X7 receptors mediate NLRP3 inflammasome-dependent IL-1β secretion in response to ATP.

    Science.gov (United States)

    Karmakar, Mausita; Katsnelson, Michael A; Dubyak, George R; Pearlman, Eric

    2016-02-15

    Although extracellular ATP is abundant at sites of inflammation, its role in activating inflammasome signalling in neutrophils is not well characterized. In the current study, we demonstrate that human and murine neutrophils express functional cell-surface P2X7R, which leads to ATP-induced loss of intracellular K(+), NLRP3 inflammasome activation and IL-1β secretion. ATP-induced P2X7R activation caused a sustained increase in intracellular [Ca(2+)], which is indicative of P2X7R channel opening. Although there are multiple polymorphic variants of P2X7R, we found that neutrophils from multiple donors express P2X7R, but with differential efficacies in ATP-induced increase in cytosolic [Ca(2+)]. Neutrophils were also the predominant P2X7R-expressing cells during Streptococcus pneumoniae corneal infection, and P2X7R was required for bacterial clearance. Given the ubiquitous presence of neutrophils and extracellular ATP in multiple inflammatory conditions, ATP-induced P2X7R activation and IL-1β secretion by neutrophils likely has a significant, wide ranging clinical impact.

  10. Galanin subtype 1 and subtype 2 receptors mediate opposite anxiety-like effects in the rat dorsal raphe nucleus.

    Science.gov (United States)

    Morais, J S; Souza, M M; Campanha, T M N; Muller, C J T; Bittencourt, A S; Bortoli, V C; Schenberg, L C; Beijamini, V

    2016-11-01

    About 40% of the dorsal raphe nucleus (DRN) neurons co-express serotonin (5-HT) and galanin. Serotonergic pathways from the DRN to the amygdala facilitate learned anxiety, while those from the DRN to the dorsal periaqueductal grey matter (DPAG) impair innate anxiety. Previously, we showed that galanin infusion in the DRN of rats induces anxiolytic effect by impairing inhibitory avoidance without changing escape behaviour in the elevated T-maze (ETM). Here, we evaluated: (1) which galanin receptors would be involved in the anxiolytic effect of galanin in the DRN of rats tested in the ETM; (2) the effects of galanin intra-DRN on panic-like behaviours evoked by electrical stimulation of the DPAG. The activation of DRN GAL1 receptors by M617 (1.0 and 3.0nmol) facilitated inhibitory avoidance, whereas the activation of GAL2 receptors by AR-M1896 (3.0nmol) impaired the inhibitory avoidance in the ETM, suggesting an anxiogenic and an anxiolytic-like effect respectively. Both agonists did not change escape behaviour in the ETM or locomotor activity in the open field. The anxiolytic effect of AR-M1896 was attenuated by the prior administration of WAY100635 (0.18nmol), a 5-HT1A antagonist. Galanin (0.3nmol) administered in the DRN increased discreetly flight behaviours induced by electrical stimulation of the DPAG, suggesting a panicolytic effect. Together, our results showed that galanin mediates opposite anxiety responses in the DRN by activation of GAL1 and GAL2 receptors. The anxiolytic effect induced by activation of Gal2 receptors may depend on serotonergic tone. Finally, the role of galanin in panic related behaviours remains uncertain. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. CD36 is not involved in scavenger receptor-mediated endocytic uptake of glycolaldehyde- and methylglyoxal-modified proteins by liver endothelial cells.

    Science.gov (United States)

    Nakajou, Keisuke; Horiuchi, Seikoh; Sakai, Masakazu; Hirata, Kenshiro; Tanaka, Makiko; Takeya, Motohiro; Kai, Toshiya; Otagiri, Masaki

    2005-05-01

    Circulating proteins modified by advanced glycation end-products (AGE) are mainly taken up by liver endothelial cells (LECs) via scavenger receptor-mediated endocytosis. Endocytic uptake of chemically modified proteins by macrophages and macrophage-derived cells is mediated by class A scavenger receptor (SR-A) and CD36. In a previous study using SR-A knockout mice, we demonstrated that SR-A is not involved in endocytic uptake of AGE proteins by LECs [Matsumoto et al. (2000) Biochem. J. 352, 233-240]. The present study was conducted to determine the contribution of CD36 to this process. Glycolaldehyde-modified BSA (GA-BSA) and methylglyoxal-modified BSA (MG-BSA) were used as AGE proteins. 125I-GA-BSA and 125I-MG-BSA underwent endocytic degradation by these cells at 37 degrees C, and this process was inhibited by several ligands for the scavenger receptors. However, this endocytic uptake of 125I-GA-BSA by LECs was not inhibited by a neutralizing anti-CD36 antibody. Similarly, hepatic uptake of (111)In-GA-BSA after its intravenous injection was not significantly attenuated by co-administration of the anti-CD36 antibody. These results clarify that CD36 does not play a significant role in elimination of GA-BSA and MG-BSA from the circulation, suggesting that the receptor involved in endocytic uptake of circulating AGE proteins by LEC is not SR-A or CD36.

  12. Heterogenous GABA(B) receptor-mediated pathways are involved in the local GABAergic system of the rat trigeminal ganglion: possible involvement of KCTD proteins.

    Science.gov (United States)

    Hayasaki, H; Sohma, Y; Kanbara, K; Otsuki, Y

    2012-08-30

    It is well known that Gamma-aminobutyric acid (GABA) plays an important role in signal transduction in the central nervous system. However, the function of GABA in the peripheral nervous system, including sensory ganglions, is still unclear. In this study we have characterized the expression, cellular distribution, and function of GABA(B) receptor subunits, and the recently discovered GABA(B) auxiliary subunits, K(+) channel tetramerization domain-containing (KCTD) proteins, in rat trigeminal ganglion (TG) neuronal cells, which are devoid of synapses. We found heterogeneous expression of both GABA(B1) and GABA(B2) subunits, and a near-plasma membrane localization of KCTD12. In addition, we found that GABA(B2) subunits correlated with KCTD16. Whole-cell current-clamp recordings showed that responses to the GABA(B) receptor agonist, baclofen, were variable and both increases and decreases in excitability were observed. This correlated with observed differences in voltage-dependent K(+) current responses to baclofen in voltage-clamped TG neuronal cells. The functional diversity of the GABA(B)ergic regulation on the excitability of the TG neuronal cell bodies could be due to the heterogenous expression of KCTD proteins, and subsequent regulation of plasma membrane K(+) channels. Taken together with our previous demonstration of a local GABA(A) receptor-mediated system in rat TG, we provide an updated GABAergic model in the rat TG that incorporates both GABA(A)- and GABA(B)-receptor systems.

  13. Electrochemical evidence for asialoglycoprotein receptor--mediated hepatocyte adhesion and proliferation in three dimensional tissue engineering scaffolds.

    Science.gov (United States)

    Vasanthan, Kirthanashri S; Sethuraman, Swaminathan; Parthasarathy, Meera

    2015-08-26

    Asialoglycoprotein receptor (ASGPR) is one of the recognition motifs on the surface of hepatocytes, which promote their adhesion to extracellular matrix in liver tissue and appropriate artificial surfaces. ASGPR-mediated adhesion is expected to minimize trans-differentiation of hepatocytes in vitro that is generally observed in integrin-mediated adhesion. The aim of the present study is to verify the role of ASGPR in hepatocyte adhesion and proliferation in scaffolds for hepatic tissue engineering. Scanning Electrochemical Microscopy (SECM) is emerging as a suitable non-invasive analytical tool due to its high sensitivity and capability to correlate the morphology and activity of live cells. HepG2 cells and rat primary hepatocytes cultured in Polyvinyl alcohol (PVA)/Gelatin hydrogel scaffolds with and without galactose (a ligand for ASGPR) modification are studied using SECM. Systematic investigation of live cells cultured for different durations in scaffolds of different compositions (9:1 and 8:2 PVA:Gelatin with and without galactose) reveals significant improvement in cell-cell communication and proliferation on galactose incorporated scaffolds, thereby demonstrating the positive influence of ASGPR-mediated adhesion. In this work, we have also developed a methodology to quantify the respiratory activity and intracellular redox activity of live cells cultured in porous tissue engineering scaffolds. Using this methodology, SECM results are compared with routine cell culture assays viz., MTS ((1-Oxyl-2,2,5,5,-tetramethyl-Δ3-pyrroline-3-methyl) Methanethiosulfonate) and Albumin assays to demonstrate the better sensitivity of SECM. In addition, the present study demonstrates SECM as a reliable and sensitive tool to monitor the activity of live cells cultured in scaffolds for tissue engineering, which could be used on a routine basis.

  14. CHD8, A Novel Beta-Catenin Associated Chromatin Remodeling Enzyme, Regulates Androgen Receptor Mediated Gene Transcription

    Science.gov (United States)

    2010-03-01

    NP- 40 , Nonidet P - 40 ; PPAR, peroxisome prolif- erator-activated receptor; PSA, prostate-specific antigen; SDS, sodium dodecyl sulfate; siRNA, small... Nonidet P - 40 (NP- 40 ). Cell lysates were cleared by centrifugation at 20,800 g for 10 min at 4 C and used for protein interaction studies as...or therapeutic target in prostate cancer. REFERENCES 1. Mulholland, D. J., Cheng, H., Reid, K., Rennie, P . S., and Nelson, C. C. (2002) J Biol

  15. Pathogenesis of Aryl Hydrocarbon Receptor-Mediated Development of Lymphoma Is Associated with Increased Cyclooxygenase-2 Expression

    OpenAIRE

    Vogel, Christoph F. A.; Li, Wen; Sciullo, Eric; Newman, John; Hammock, Bruce; Reader, J. Rachel; Tuscano, Joseph; Matsumura, Fumio

    2007-01-01

    Epidemiological studies indicate that exposure to environmental pollutants such as pesticides and dioxins leads to the pathogenesis of lymphoma and leukemia. Here, we show that activation of the aryl hydrocarbon receptor (AhR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in loss of the programmed cell death (apoptosis) response in three different lymphoma cell lines, which plays a key role in the development of cancer, especially lymphoma and leukemia. The AhR-mediated inhibition of...

  16. The Stimulus Effects of 8-OH-DPAT: Evidence for a 5-HT2A Receptor-Mediated Component

    OpenAIRE

    Reissig, C.J.; Eckler, J.R.; Rabin, R. A.; Rice, K. C.; Winter, J. C.

    2007-01-01

    A previous investigation in our laboratory found that the stimulus effects of the 5-HT2A agonist, LSD, are potentiated by 5-HT1A receptor agonists including the prototypic agonist, 8-OH-DPAT. Also suggestive of behaviorally relevant interactions between 5-HT1A and 5-HT2A receptors are behavioral analyses of locomotor activity, head twitch response, forepaw treading and production of the serotonin syndrome; in some instances effects are augmented, in other, diminished. These observations led u...

  17. Enhancement of NMDA receptor-mediated excitatory postsynaptic currents by gp120-treated macrophages: Implications for HIV-1-associated neuropathology

    OpenAIRE

    Yang, Jianming; Hu, Dehui; Xia, Jianxun; Liu, Jianuo; Zhang, Gang; Gendelman, Howard E; Nawal M. Boukli; Xiong, Huangui

    2013-01-01

    A plethora of prior studies has linked HIV-1-infected and immune activated brain mononuclear phagocytes (MP; blood borne macrophages and microglia) to neuronal dysfunction. These are modulated by N-methyl-D-aspartate receptor (NMDAR) antagonists and supporting their relevance for HIV-1-associated nervous system disease. The role of NMDAR subsets in HIV-1-induced neuronal injury, nonetheless, is poorly understood. To this end, we investigated conditioned media from HIV-1gp120-treated human mon...

  18. EGF receptor signaling blocks aryl hydrocarbon receptor-mediated transcription and cell differentiation in human epidermal keratinocytes

    OpenAIRE

    Sutter, Carrie Hayes; Yin, Hong; Li, Yunbo; Mammen, Jennifer S.; Bodreddigari, Sridevi; Stevens, Gaylene; Cole, Judith A; Sutter, Thomas R.

    2009-01-01

    Dioxin is an extremely potent carcinogen. In highly exposed people, the most commonly observed toxicity is chloracne, a pathological response of the skin. Most of the effects of dioxin are attributed to its activation of the aryl hydrocarbon receptor (AHR), a transcription factor that binds to the Ah receptor nuclear translocator (ARNT) to regulate the transcription of numerous genes, including CYP1A1 and CYP1B1. In cultures of normal human epidermal keratinocytes dioxin accelerates cell diff...

  19. Molecular mechanisms underlying β-adrenergic receptor-mediated cross-talk between sympathetic neurons and immune cells.

    Science.gov (United States)

    Lorton, Dianne; Bellinger, Denise L

    2015-03-11

    Cross-talk between the sympathetic nervous system (SNS) and immune system is vital for health and well-being. Infection, tissue injury and inflammation raise firing rates of sympathetic nerves, increasing their release of norepinephrine (NE) in lymphoid organs and tissues. NE stimulation of β2-adrenergic receptors (ARs) in immune cells activates the cAMP-protein kinase A (PKA) intracellular signaling pathway, a pathway that interfaces with other signaling pathways that regulate proliferation, differentiation, maturation and effector functions in immune cells. Immune-SNS cross-talk is required to maintain homeostasis under normal conditions, to develop an immune response of appropriate magnitude after injury or immune challenge, and subsequently restore homeostasis. Typically, β2-AR-induced cAMP is immunosuppressive. However, many studies report actions of β2-AR stimulation in immune cells that are inconsistent with typical cAMP-PKA signal transduction. Research during the last decade in non-immune organs, has unveiled novel alternative signaling mechanisms induced by β2-AR activation, such as a signaling switch from cAMP-PKA to mitogen-activated protein kinase (MAPK) pathways. If alternative signaling occurs in immune cells, it may explain inconsistent findings of sympathetic regulation of immune function. Here, we review β2-AR signaling, assess the available evidence for alternative signaling in immune cells, and provide insight into the circumstances necessary for "signal switching" in immune cells.

  20. α7 Nicotinic receptor-mediated astrocytic gliotransmitter release: Aβ effects in a preclinical Alzheimer's mouse model.

    Directory of Open Access Journals (Sweden)

    Tiina Maria Pirttimaki

    Full Text Available It is now recognized that astrocytes participate in synaptic communication through intimate interactions with neurons. A principal mechanism is through the release of gliotransmitters (GTs such as ATP, D-serine and most notably, glutamate, in response to astrocytic calcium elevations. We and others have shown that amyloid-β (Aβ, the toxic trigger for Alzheimer's disease (AD, interacts with hippocampal α7 nicotinic acetylcholine receptors (nAChRs. Since α7nAChRs are highly permeable to calcium and are expressed on hippocampal astrocytes, we investigated whether Aβ could activate astrocytic α7nAChRs in hippocampal slices and induce GT glutamate release. We found that biologically-relevant concentrations of Aβ1-42 elicited α7nAChR-dependent calcium elevations in hippocampal CA1 astrocytes and induced NMDAR-mediated slow inward currents (SICs in CA1 neurons. In the Tg2576 AD mouse model for Aβ over-production and accumulation, we found that spontaneous astrocytic calcium elevations were of higher frequency compared to wildtype (WT. The frequency and kinetic parameters of AD mice SICs indicated enhanced gliotransmission, possibly due to increased endogenous Aβ observed in this model. Activation of α7nAChRs on WT astrocytes increased spontaneous inward currents on pyramidal neurons while α7nAChRs on astrocytes of AD mice were abrogated. These findings suggest that, at an age that far precedes the emergence of cognitive deficits and plaque deposition, this mouse model for AD-like amyloidosis exhibits augmented astrocytic activity and glutamate GT release suggesting possible repercussions for preclinical AD hippocampal neural networks that contribute to subsequent cognitive decline.

  1. Kinin B1 receptors mediate depression-like behavior response in stressed mice treated with systemic E. coli lipopolysaccharide

    Directory of Open Access Journals (Sweden)

    Campos Maria M

    2010-12-01

    Full Text Available Abstract Background Kinin B1 receptors are inducible molecules up-regulated after inflammatory stimuli. This study evaluated the relevance of kinin B1 receptors in a mouse depression behavior model. Methods Mice were exposed to a 5-min swimming session, and 30 min later they were injected with E. coli lipopolysaccharide (LPS. Depression-like behavior was assessed by determining immobility time in a tail suspension test. Different brain structures were collected for molecular and immunohistochemical studies. Anhedonia was assessed by means of a sucrose intake test. Results Our protocol elicited an increase in depression-like behavior in CF1 mice, as assessed by the tail-suspension test, at 24 h. This behavior was significantly reduced by treatment with the selective B1 receptor antagonists R-715 and SSR240612. Administration of SSR240612 also prevented an increase in number of activated microglial cells in mouse hippocampus, but did not affect a reduction in expression of mRNA for brain-derived neurotrophic factor. The increased immobility time following LPS treatment was preceded by an enhancement of hippocampal and cortical B1 receptor mRNA expression (which were maximal at 1 h, and a marked production of TNFα in serum, brain and cerebrospinal fluid (between 1 and 6 h. The depression-like behavior was virtually abolished in TNFα p55 receptor-knockout mice, and increased B1 receptor mRNA expression was completely absent in this mouse strain. Furthermore, treatment with SSR240612 was also effective in preventing anhedonia in LPS-treated mice, as assessed using a sucrose preference test. Conclusion Our data show, for the first time, involvement of kinin B1 receptors in depressive behavioral responses, in a process likely associated with microglial activation and TNFα production. Thus, selective and orally active B1 receptor antagonists might well represent promising pharmacological tools for depression therapy.

  2. RhoA and Rac1 GTPases Differentially Regulate Agonist-Receptor Mediated Reactive Oxygen Species Generation in Platelets

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    Akbar, Huzoor; Duan, Xin; Saleem, Saima; Davis, Ashley K.; Zheng, Yi

    2016-01-01

    Agonist induced generation of reactive oxygen species (ROS) by NADPH oxidases (NOX) enhances platelet aggregation and hence the risk of thrombosis. RhoA and Rac1 GTPases are involved in ROS generation by NOX in a variety of cells, but their roles in platelet ROS production remain unclear. In this study we used platelets from RhoA and Rac1 conditional knockout mice as well as human platelets treated with Rhosin and NSC23767, rationally designed small molecule inhibitors of RhoA and Rac GTPases, respectively, to better define the contributions of RhoA and Rac1 signaling to ROS generation and platelet activation. Treatment of platelets with Rhosin inhibited: (a) U46619 induced activation of RhoA; (b) phosphorylation of p47phox, a critical component of NOX; (c) U46619 or thrombin induced ROS generation; (d) phosphorylation of myosin light chain (MLC); (e) platelet shape change; (f) platelet spreading on immobilized fibrinogen; and (g) release of P-selectin, secretion of ATP and aggregation. Conditional deletion of RhoA or Rac1 gene inhibited thrombin induced ROS generation in platelets. Addition of Y27632, a RhoA inhibitor, NSC23766 or Phox-I, an inhibitor of Rac1-p67phox interaction, to human platelets blocked thrombin induced ROS generation. These data suggest that: (a) RhoA/ROCK/p47phox signaling axis promotes ROS production that, at least in part, contributes to platelet activation in conjunction with or independent of the RhoA/ROCK mediated phosphorylation of MLC; and (b) RhoA and Rac1 differentially regulate ROS generation by inhibiting phosphorylation of p47phox and Rac1-p67phox interaction, respectively. PMID:27681226

  3. Receptor subtype involved in α1-adrenergic receptor-mediated Ca2+ sig-naling in cardiomyocytes

    Institute of Scientific and Technical Information of China (English)

    Da-li LUO; Jian GAO; Lin-lin FAN; Yu TANG; You-yi ZHANG; Qi-de HAN

    2007-01-01

    Aim: The enhancement of intracellular Ca2+ signaling in response to α1-adrener-gic receptor (α1-AR) stimulation is an essential signal transduction event in the regulation of cardiac functions, such as cardiac growth, cardiac contraction, and cardiac adaptation to various situations. The present study was intended to determine the role(s) of the α1-AR subtype(s) in mediating this response. Methods: We evaluated the effects of subtype-specific agonists and antagonists of the α1- AR on the intracellular Ca2+ signaling of neonatal rat ventricular myocytes using a confocal microscope. Results: After being cultured for 48 h, the myocytes exhibited spontaneous local Ca2+ release, sparks, and global Ca2+ transients. The activation of the α1-AR with phenylephrine, a selective agonist of the α1-AR, dose-dependently increased the frequency of Ca2+ transients with an EC50 value of 2.3 μmol/L. Blocking the α1A-AR subtype with 5-methyhirapidil (5-Mu) inhi-bited the stimulatory effect of phenylephrine with an IC50 value of 6.7 nmol/L. In contrast, blockade of the α1B-AR and α1D-AR subtypes with chloroethylclonidine and BMY 7378, respectively, did not affect the phenylephrine effect. Similarly, the local Ca2+ spark numbers were also increased by the activation of theα1-AR, and this effect could be abolished selectively by 5-Mu. More importantly, A61603, a novel selective α1A-AR agonist, mimicked the effects of phenylephrine, but with more potency (EC50 value =6.9 nmol/L) in the potentiation of Ca2+ transients, and blockade of the α1A-AR by 5-Mu caused abolishment of its effects. Conclusion: These results indicate that α1-adrenergic stimulation of intracellular Ca2+ activity is mediated selectively by the α1A-AR.

  4. Leukotriene B4 modulates P2X7 receptor-mediated Leishmania amazonensis elimination in murine macrophages.

    Science.gov (United States)

    Chaves, Mariana M; Marques-da-Silva, Camila; Monteiro, Ana Paula T; Canetti, Cláudio; Coutinho-Silva, Robson

    2014-05-15

    ATP is an important signaling molecule in the immune system, and it is able to bind the P2X7 purinergic receptor. Recently, our group showed that ATP-treated macrophages eliminate Leishmania amazonensis. It has been reported that leukotriene B4 (LTB4) reduces the parasitic load of infected macrophages. Additionally, it has been demonstrated that the P2X7 receptor can induce PLA2 activation and arachidonic acid mobilization. Based on these findings, we investigated whether LTB4 is produced upon P2X7 receptor activation and examined whether LTB4 modulates parasite elimination. Using macrophages lacking the P2X7 receptor, we observed that ATP was not able to reduce L. amazonensis load. This result suggests a role of the P2X7 purinergic receptor in parasite elimination. In addition, ATP was sufficient to induce LTB4 release from infected control macrophages but not from macrophages lacking the P2X7 receptor. Moreover, we found that ATP failed to decrease the parasitic load in 5-lipoxygenase (LO)-deficient macrophages. Treatment with the 5-LO inhibitor AA861 also impairs the ATP effect on parasitic loads. Furthermore, macrophages from 5-LO knockout mice eliminated L. amazonensis in the presence of exogenous LTB4, and macrophages obtained from P2X7 receptor knockout mice eliminated L. amazonensis when incubated with ionomycin. Finally, we demonstrated that in the presence of CP105696, an antagonist for LTB4 high-affinity receptor, ATP was not able to reduce parasitic load. These results indicate that P2X7 receptor activation leads to LTB4 formation, which is required for L. amazonensis elimination.

  5. Food-associated estrogenic compounds induce estrogen receptor-mediated luciferase gene expression in transgenic male mice.

    Science.gov (United States)

    Ter Veld, Marcel G R; Zawadzka, E; van den Berg, J H J; van der Saag, Paul T; Rietjens, Ivonne M C M; Murk, Albertinka J

    2008-07-30

    The present paper aims at clarifying to what extent seven food-associated compounds, shown before to be estrogenic in vitro, can induce estrogenic effects in male mice with an estrogen receptor (ER)-mediated luciferase (luc) reporter gene system. The luc induction was determined in different tissues 8h after dosing the ER-luc male mice intraperitoneally (IP) or 14h after oral dosing. Estradiol-propionate (EP) was used as a positive control at 0.3 and 1mg/kg bodyweight (bw), DMSO as solvent control. The food-associated estrogenic compounds tested at non-toxic doses were bisphenol A (BPA) and nonylphenol (NP) (both at 10 and 50mg/kgbw), dichlorodiphenyldichloroethylene (p,p'-DDE; at 5 and 25mg/kgbw), quercetin (at 1.66 and 16.6mg/kgbw), di-isoheptyl phthalate (DIHP), di-(2-ethylhexyl) phthalate (DEHP) and di-(2-ethylhexyl) adipate (DEHA) all at 30 and 100mg/kgbw. In general IP dosing resulted in higher luc inductions than oral dosing. EP induced luc activity in the liver in a statistically significant dose-related way with the highest induction of all compounds tested which was 20,000 times higher than the induction by the DMSO-control. NP, DDE, DEHA and DIHP did not induce luc activity in any of the tissues tested. BPA induced luc in the liver up to 420 times via both exposure routes. BPA, DEHP and quercetin induced luc activity in the liver after oral exposure. BPA (50mg/kgbw IP) also induced luc activity in the testis, kidneys and tibia. The current study reveals that biomarker-responses in ER-luc male mice occur after a single oral exposure to food-associated estrogenic model compounds at exposure levels 10 to 10(4) times higher than the established TDI's for some of these compounds. Given the facts that (i) the present study did not include chronic exposure and that (ii) simultaneous exposure to multiple estrogenic compounds may be a realistic exposure scenario, it remains to be seen whether this margin is sufficiently high.

  6. Effects of some new antidepressant drugs on the glucocorticoid receptor-mediated gene transcription in fibroblast cells.

    Science.gov (United States)

    Augustyn, Matylda; Otczyk, Magdalena; Budziszewska, Bogusława; Jagła, Grzegorz; Nowak, Wojciech; Basta-Kaim, Agnieszka; Jaworska-Feil, Lucylla; Kubera, Marta; Tetich, Magdalena; Leśkiewicz, Monika; Lasoń, Władysław

    2005-01-01

    Antidepressant drugs are thought to counteract effects of hypercortisolemia, frequently associated with depression, by lowering cortisol level and by modifying the function of glucocorticoid receptors (GR). Indeed, classical antidepressants inhibit corticosteroid-induced gene transcription in cell cultures. The aim of the present study was to investigate effects of new generation antidepressant drugs on GR function in mouse fibroblast cells (L929), stably transfected with mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) plasmid (LMCAT cells). It has been found that reboxetine (at 10 and 30 microM), venlafaxine, citalopram and mirtazapine (at 30 microM), but not milnacipran, in statistically significant manner inhibited corticosterone-induced gene transcription. However, the effects of new generation antidepressant drugs were weaker than those evoked by imipramine, which was active already at 3 microM concentration. Further studies on the mechanism of antidepressant action on GR function revealed that protein kinase C, but not mitogen-activated protein kinases (MAPK), glycogen synthase kinase (GSK-3) and protein kinase B (PKB, Akt) play a role in this phenomenon.

  7. Pre-synaptic adenosine A2A receptors control cannabinoid CB1 receptor-mediated inhibition of striatal glutamatergic neurotransmission.

    Science.gov (United States)

    Martire, Alberto; Tebano, Maria Teresa; Chiodi, Valentina; Ferreira, Samira G; Cunha, Rodrigo A; Köfalvi, Attila; Popoli, Patrizia

    2011-01-01

    An interaction between adenosine A(2A) receptors (A(2A) Rs) and cannabinoid CB(1) receptors (CB(1) Rs) has been consistently reported to occur in the striatum, although the precise mechanisms are not completely understood. As both receptors control striatal glutamatergic transmission, we now probed the putative interaction between pre-synaptic CB(1) R and A(2A) R in the striatum. In extracellular field potentials recordings in corticostriatal slices from Wistar rats, A(2A) R activation by CGS21680 inhibited CB(1) R-mediated effects (depression of synaptic response and increase in paired-pulse facilitation). Moreover, in superfused rat striatal nerve terminals, A(2A) R activation prevented, while A(2A) R inhibition facilitated, the CB(1) R-mediated inhibition of 4-aminopyridine-evoked glutamate release. In summary, the present study provides converging neurochemical and electrophysiological support for the occurrence of a tight control of CB(1) R function by A(2A) Rs in glutamatergic terminals of the striatum. In view of the key role of glutamate to trigger the recruitment of striatal circuits, this pre-synaptic interaction between CB(1) R and A(2A) R may be of relevance for the pathogenesis and the treatment of neuropsychiatric disorders affecting the basal ganglia.

  8. Toll-Like Receptor Mediated Modulation of T Cell Response by Commensal Intestinal Microbiota as a Trigger for Autoimmune Arthritis

    Directory of Open Access Journals (Sweden)

    Rebecca Rogier

    2015-01-01

    Full Text Available In autoimmune diseases, a disturbance of the balance between T helper 17 (Th17 and regulatory T cells (Tregs is often observed. This disturbed balance is also the case in rheumatoid arthritis (RA. Genetic predisposition to RA confers the presence of several polymorphisms mainly regulating activation of T lymphocytes. However, the presence of susceptibility factors is neither necessary nor sufficient to explain the disease development, emphasizing the importance of environmental factors. Multiple studies have shown that commensal gut microbiota is of great influence on immune homeostasis and can trigger the development of autoimmune diseases by favoring induction of Th17 cells over Tregs. However the mechanism by which intestinal microbiota influences the Th cell balance is not completely understood. Here we review the current evidence supporting the involvement of commensal intestinal microbiota in rheumatoid arthritis, along with a potential role of Toll-like receptors (TLRs in modulating the relevant Th cell responses to trigger autoimmunity. A better understanding of TLR triggering by intestinal microbiota and subsequent T cell activation might offer new perspectives for manipulating the T cell response in RA patients and may lead to the discovery of new therapeutic targets or even preventive measures.

  9. Neurokinin 1 receptor mediates membrane blebbing and sheer stress-induced microparticle formation in HEK293 cells.

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    Panpan Chen

    Full Text Available Cell-derived microparticles participate in intercellular communication similar to the classical messenger systems of small and macro-molecules that bind to specialized membrane receptors. Microparticles have been implicated in the regulation of a variety of complex physiopathologic processes, such as thrombosis, the control of innate and adaptive immunity, and cancer. The neurokinin 1 receptor (NK1R is a Gq-coupled receptor present on the membrane of a variety of tissues, including neurons in the central and peripheral nervous system, immune cells, endocrine and exocrine glands, and smooth muscle. The endogenous agonist of NK1R is the undecapeptide substance P (SP. We have previously described intracellular signaling mechanisms that regulate NK1R-mediated rapid cell shape changes in HEK293 cells and U373MG cells. In the present study, we show that the activation of NK1R in HEK293 cells, but not in U373MG cells, leads to formation of sheer-stress induced microparticles that stain positive with the membrane-selective fluorescent dye FM 2-10. SP-induced microparticle formation is independent of elevated intracellular calcium concentrations and activation of NK1R present on HEK293-derived microparticles triggers detectable calcium increase in SP-induced microparticles. The ROCK inhibitor Y27632 and the dynamin inhibitor dynasore inhibited membrane blebbing and microparticle formation in HEK293 cells, strongly suggesting that microparticle formation in this cell type is dependent on membrane blebbing.

  10. Delta-opioid receptors mediate unique plasticity onto parvalbumin-expressing interneurons in area CA2 of the hippocampus.

    Science.gov (United States)

    Piskorowski, Rebecca A; Chevaleyre, Vivien

    2013-09-04

    Inhibition is critical for controlling information transfer in the brain. However, the understanding of the plasticity and particular function of different interneuron subtypes is just emerging. Using acute hippocampal slices prepared from adult mice, we report that in area CA2 of the hippocampus, a powerful inhibitory transmission is acting as a gate to prevent CA3 inputs from driving CA2 neurons. Furthermore, this inhibition is highly plastic, and undergoes a long-term depression following high-frequency 10 Hz or theta-burst induction protocols. We describe a novel form of long-term depression at parvalbumin-expressing (PV+) interneuron synapses that is dependent on delta-opioid receptor (DOR) activation. Additionally, PV+ interneuron transmission is persistently depressed by DOR activation in area CA2 but only transiently depressed in area CA1. These results provide evidence for a differential temporal modulation of PV+ synapses between two adjacent cortical circuits, and highlight a new function of PV+ cells in controlling information transfer.

  11. Archaeosomes varying in lipid composition differ in receptor-mediated endocytosis and differentially adjuvant immune responses to entrapped antigen

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    G. Dennis Sprott

    2003-01-01

    Full Text Available Archaeosomes prepared from total polar lipids extracted from six archaeal species with divergent lipid compositions had the capacity to deliver antigen for presentation via both MHC class I and class II pathways. Lipid extracts from Halobacterium halobium and from Halococcus morrhuae strains 14039 and 16008 contained archaetidylglycerol methylphosphate and sulfated glycolipids rich in mannose residues, and lacked archaetidylserine, whereas the opposite was found in Methanobrevibacter smithii, Methanosarcina mazei and Methanococcus jannaschii. Annexin V labeling revealed a surface orientation of phosphoserine head groups in M. smithii, M. mazei and M. jannaschii archaeosomes. Uptake of rhodamine-labeled M. smithii or M. jannaschii archaeosomes by murine peritoneal macrophages was inhibited by unlabeled liposomes containing phosphatidylserine, by the sulfhydryl inhibitor N-ethylmaleimide, and by ATP depletion using azide plus fluoride, but not by H. halobium archaeosomes. In contrast, N-ethylmaleimide failed to inhibit uptake of the four other rhodamine-labeled archaeosome types, and azide plus fluoride did not inhibit uptake of H. halobium or H. morrhuae archaeosomes. These results suggest endocytosis of archaeosomes rich in surface-exposed phosphoserine head groups via a phosphatidylserine receptor, and energy-independent surface adsorption of certain other archaeosome composition classes. Lipid composition affected not only the endocytic mechanism, but also served to differentially modulate the activation of dendritic cells. The induction of IL-12 secretion from dendritic cells exposed to H. morrhuae 14039 archaeosomes was striking compared with cells exposed to archaeosomes from 16008. Thus, archaeosome types uniquely modulate antigen delivery and dendritic cell activation.

  12. Androgen Receptor-Mediated Growth Suppression of HPr-1AR and PC3-Lenti-AR Prostate Epithelial Cells.

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    Young-Chae Kim

    Full Text Available The androgen receptor (AR mediates the developmental, physiologic, and pathologic effects of androgens including 5α-dihydrotestosterone (DHT. However, the mechanisms whereby AR regulates growth suppression and differentiation of luminal epithelial cells in the prostate gland and proliferation of malignant versions of these cells are not well understood, though they are central to prostate development, homeostasis, and neoplasia. Here, we identify androgen-responsive genes that restrain cell cycle progression and proliferation of human prostate epithelial cell lines (HPr-1AR and PC3-Lenti-AR, and we investigate the mechanisms through which AR regulates their expression. DHT inhibited proliferation of HPr-1AR and PC3-Lenti-AR, and cell cycle analysis revealed a prolonged G1 interval. In the cell cycle, the G1/S-phase transition is initiated by the activity of cyclin D and cyclin-dependent kinase (CDK complexes, which relieve growth suppression. In HPr-1AR, cyclin D1/2 and CDK4/6 mRNAs were androgen-repressed, whereas CDK inhibitor, CDKN1A, mRNA was androgen-induced. The regulation of these transcripts was AR-dependent, and involved multiple mechanisms. Similar AR-mediated down-regulation of CDK4/6 mRNAs and up-regulation of CDKN1A mRNA occurred in PC3-Lenti-AR. Further, CDK4/6 overexpression suppressed DHT-inhibited cell cycle progression and proliferation of HPr-1AR and PC3-Lenti-AR, whereas CDKN1A overexpression induced cell cycle arrest. We therefore propose that AR-mediated growth suppression of HPr-1AR involves cyclin D1 mRNA decay, transcriptional repression of cyclin D2 and CDK4/6, and transcriptional activation of CDKN1A, which serve to decrease CDK4/6 activity. AR-mediated inhibition of PC3-Lenti-AR proliferation occurs through a similar mechanism, albeit without down-regulation of cyclin D. Our findings provide insight into AR-mediated regulation of prostate epithelial cell proliferation.

  13. Age-related reduction in estrogen receptor-mediated mechanisms of vascular relaxation in female spontaneously hypertensive rats.

    Science.gov (United States)

    Wynne, Fanisha L; Payne, Jason A; Cain, Ashley E; Reckelhoff, Jane F; Khalil, Raouf A

    2004-02-01

    Hypertension increases with aging, and changes in vascular estrogen receptors (ERs) may play a role in age-related hypertension in women. We tested whether age-related increases in blood pressure in female spontaneously hypertensive rats (SHRs) are associated with reduction in amount and/or vascular relaxation effects of estrogen and ER. Arterial pressure and plasma estradiol were measured in adult (12 weeks) and aging (16 months) female SHRs, and thoracic aorta was isolated for measurement of active stress, 45Ca2+ influx, and ERs. Arterial pressure was greater and plasma estradiol was less in aging females than in adult females. In aorta of adult females, Western blots revealed alpha- and beta-ERs that were slightly reduced in aging rats. In endothelium-intact vascular strips, phenylephrine (Phe; 10(-5) mol/L) caused greater active stress in aging rats (9.3+/-0.2) than in adult rats (6.2+/-0.3x10(4) N/m2). 17beta-estradiol (E2) caused relaxation of Phe contraction and stimulation of vascular nitrite/nitrate production, which was reduced in aging rats. In endothelium-denuded strips, E2 still caused relaxation of Phe contraction, which was smaller in aging rats than adult rats. KCl (51 mmol/L), which stimulates Ca2+ influx, produced greater active stress in aging rats (9.1+/-0.3) than in adult rats (5.9+/-0.2x10(4) N/m2). E2 caused relaxation of KCl contraction and inhibition of Phe- and KCl-induced 45Ca2+ influx, which were reduced in aging rats. Thus, aging in female SHR is associated with reduction in ER-mediated NO production from endothelial cells and decrease in inhibitory effects of estrogen on Ca2+ entry mechanisms of smooth muscle contraction. The age-related decrease in ER-mediated vascular relaxation may explain the increased vascular contraction and arterial pressure associated with aging in females.

  14. Presynaptic Adenosine Receptor-Mediated Regulation of Diverse Thalamocortical Short-Term Plasticity in the Mouse Whisker Pathway.

    Science.gov (United States)

    Ferrati, Giovanni; Martini, Francisco J; Maravall, Miguel

    2016-01-01

    Short-term synaptic plasticity (STP) sets the sensitivity of a synapse to incoming activity and determines the temporal patterns that it best transmits. In "driver" thalamocortical (TC) synaptic populations, STP is dominated by depression during stimulation from rest. However, during ongoing stimulation, lemniscal TC connections onto layer 4 neurons in mouse barrel cortex express variable STP. Each synapse responds to input trains with a distinct pattern of depression or facilitation around its mean steady-state response. As a result, in common with other synaptic populations, lemniscal TC synapses express diverse rather than uniform dynamics, allowing for a rich representation of temporally varying stimuli. Here, we show that this STP diversity is regulated presynaptically. Presynaptic adenosine receptors of the A1R type, but not kainate receptors (KARs), modulate STP behavior. Blocking the receptors does not eliminate diversity, indicating that diversity is related to heterogeneous expression of multiple mechanisms in the pathway from presynaptic calcium influx to neurotransmitter release.

  15. Presynaptic adenosine receptor-mediated regulation of diverse thalamocortical short-term plasticity in the mouse whisker pathway

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    Giovanni eFerrati

    2016-02-01

    Full Text Available Short-term synaptic plasticity (STP sets the sensitivity of a synapse to incoming activity and determines the temporal patterns that it best transmits. In driver thalamocortical (TC synaptic populations, STP is dominated by depression during stimulation from rest. However, during ongoing stimulation, lemniscal TC connections onto layer 4 neurons in mouse barrel cortex express variable STP. Each synapse responds to input trains with a distinct pattern of depression or facilitation around its mean steady-state response. As a result, in common with other synaptic populations, lemniscal TC synapses express diverse rather than uniform dynamics, allowing for a rich representation of temporally varying stimuli. Here we show that this STP diversity is regulated presynaptically. Presynaptic adenosine receptors of the A1R type, but not kainate receptors, modulate STP behavior. Blocking the receptors does not eliminate diversity, indicating that diversity is related to heterogeneous expression of multiple mechanisms in the pathway from presynaptic calcium influx to neurotransmitter release.

  16. Catecholamine stress alters neutrophil trafficking and impairs wound healing by β2-adrenergic receptor-mediated upregulation of IL-6.

    Science.gov (United States)

    Kim, Min-Ho; Gorouhi, Farzam; Ramirez, Sandra; Granick, Jennifer L; Byrne, Barbara A; Soulika, Athena M; Simon, Scott I; Isseroff, R Rivkah

    2014-03-01

    Stress-induced hormones can alter the inflammatory response to tissue injury; however, the precise mechanism by which epinephrine influences inflammatory response and wound healing is not well defined. Here we demonstrate that epinephrine alters the neutrophil (polymorphonuclear leukocyte (PMN))-dependent inflammatory response to a cutaneous wound. Using noninvasive real-time imaging of genetically tagged PMNs in a murine skin wound, chronic, epinephrine-mediated stress was modeled by sustained delivery of epinephrine. Prolonged systemic exposure of epinephrine resulted in persistent PMN trafficking to the wound site via an IL-6-mediated mechanism, and this in turn impaired wound repair. Further, we demonstrate that β2-adrenergic receptor-dependent activation of proinflammatory macrophages is critical for epinephrine-mediated IL-6 production. This study expands our current understanding of stress hormone-mediated impairment of wound healing and provides an important mechanistic link to explain how epinephrine stress exacerbates inflammation via increased number and lifetime of PMNs.

  17. The MHC class I binding proteins LIR-1 and LIR-2 inhibit Fc receptor-mediated signaling in monocytes.

    Science.gov (United States)

    Fanger, N A; Cosman, D; Peterson, L; Braddy, S C; Maliszewski, C R; Borges, L

    1998-11-01

    The MHC class I binding proteins leukocyte immunoglobulin-like receptor (LIR)-1 and -2 recognize a similar broad spectrum of HLA-A, -B and -C alleles but are differentially expressed in lymphocytes, monocytes, and dendritic cells. In monocytes, phosphorylation of LIR-1 and LIR-2 results in the binding of the tyrosine phosphatase SHP-1. Coligation of either LIR with Fcgamma receptor I (CD64) inhibits tyrosine phosphorylation of the associated Fc receptor gamma chain and Syk molecules, as well as intracellular calcium mobilization. These findings suggest that LIR-1 and LIR-2 function as unique MHC class I receptors involved in the inhibition or down-modulation of monocyte activation signals, particularly those mediated through the receptors for IgG, IgE and IgA.

  18. Adenosine A1 receptor-mediated transactivation of the EGF receptor produces a neuroprotective effect on cortical neurons in vitro

    Institute of Scientific and Technical Information of China (English)

    Ke-qiang XIE; Li-min ZHANG; Yan CAO; Jun ZHU; Lin-yin FENG

    2009-01-01

    Aim:To understand the mechanism of the transactivation of the epidermal growth factor receptor (EGFR) mediated by the adenosine A1 receptor (A1R).Methods:Primary cultured rat cortical neurons subjected to oxygen-glucose deprivation (OGD) and HEK293/A1R cells were treated with the A1R-specific agonist N6-cyclopentyladenosine (CPA).Phospho-EGFR,Akt,and ERK1/2 were observed by Western blot.An interaction between EGFR and AIR was detected using immunoprecipitation and immunocytochemistry.Results:The A1R agonist CPA causes protein kinase B (Akt) activation and protects primary cortical neurons from oxygen-glucose deprivation (OGD) insult.A1R and EGFR co-localize in the membranes of neurons and form an immunocomplex.A1R stimulation induces significant EGFR phosphorylation via a P13K and Src kinase signaling pathway;this stimulation provides a neuroprotective effect in cortical neurons.CPA leads to sustained phosphorylation of extracellularly regulated kinases 1 and 2 (ERK1/2) in cortical neurons,but only to transient phosphorylation in HEK 293/A1R cells.The response to the AtR agonist is mediated primarily through EGFR trans-activation that is dependent on pertussis toxin (PTX)-sensitive G1 protein and metalloproteases in HEK 293/A1R.Conclusion:A1R-mediated EGFR transactivation confers a neuroprotective effect in primary cortical neurons.P13 kinase and Src kinase play pivotal roles in this response.

  19. Sentrin/SUMO specific proteases as novel tissue-selective modulators of vitamin D receptor-mediated signaling.

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    Wai-Ping Lee

    Full Text Available Vitamin D receptor (VDR is a substrate for modification with small ubiquitin-like modifier (SUMO. To further assess the role of reversible SUMOylation within the vitamin D hormonal response, we evaluated the effects of sentrin/SUMO-specific proteases (SENPs that can function to remove small ubiquitin-like modifier (SUMO from target proteins upon the activities of VDR and related receptors. We report that SENP1 and SENP2 strikingly potentiate ligand-mediated transactivation of VDR and also its heterodimeric partner, retinoid X receptor (RXRα with depletion of cellular SENP1 significantly diminishing the hormonal responsiveness of the endogenous vitamin D target gene CYP24A1. We find that SENP-directed modulation of VDR activity is cell line-dependent, achieving potent modulatory effects in Caco-2 and HEK-293 cells, while in MCF-7 cells the vitamin D signal is unaffected by any tested SENP. In support of their function as novel modulators of the vitamin D hormonal pathway we demonstrate that both SENP1 and SENP2 can interact with VDR and reverse its modification with SUMO2. In a preliminary analysis we identify lysine 91, a residue known to be critical for formation and DNA binding of the VDR-RXR heterodimer, as a minor SUMO acceptor site within VDR. In combination, our results support a repressor function for SUMOylation of VDR and reveal SENPs as a novel class of VDR/RXR co-regulatory protein that significantly modulate the vitamin D response and which could also have important impact upon the functionality of both RXR-containing homo and heterodimers.

  20. Pharmacological inhibition of microsomal prostaglandin E synthase-1 suppresses epidermal growth factor receptor-mediated tumor growth and angiogenesis.

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    Federica Finetti

    Full Text Available BACKGROUND: Blockade of Prostaglandin (PG E(2 production via deletion of microsomal Prostaglandin E synthase-1 (mPGES-1 gene reduces tumor cell proliferation in vitro and in vivo on xenograft tumors. So far the therapeutic potential of the pharmacological inhibition of mPGES-1 has not been elucidated. PGE(2 promotes epithelial tumor progression via multiple signaling pathways including the epidermal growth factor receptor (EGFR signaling pathway. METHODOLOGY/PRINCIPAL FINDINGS: Here we evaluated the antitumor activity of AF3485, a compound of a novel family of human mPGES-1 inhibitors, in vitro and in vivo, in mice bearing human A431 xenografts overexpressing EGFR. Treatment of the human cell line A431 with interleukin-1beta (IL-1β increased mPGES-1 expression, PGE(2 production and induced EGFR phosphorylation, and vascular endothelial growth factor (VEGF and fibroblast growth factor-2 (FGF-2 expression. AF3485 reduced PGE(2 production, both in quiescent and in cells stimulated by IL-1β. AF3485 abolished IL-1β-induced activation of the EGFR, decreasing VEGF and FGF-2 expression, and tumor-mediated endothelial tube formation. In vivo, in A431 xenograft, AF3485, administered sub-chronically, decreased tumor growth, an effect related to inhibition of EGFR signalling, and to tumor microvessel rarefaction. In fact, we observed a decrease of EGFR phosphorylation, and VEGF and FGF-2 expression in tumours explanted from treated mice. CONCLUSION: Our work demonstrates that the pharmacological inhibition of mPGES-1 reduces squamous carcinoma growth by suppressing PGE(2 mediated-EGFR signalling and by impairing tumor associated angiogenesis. These results underscore the potential of mPGES-1 inhibitors as agents capable of controlling tumor growth.

  1. Role of heme oxygenase 1 in TNF/TNF receptor-mediated apoptosis after hepatic ischemia/reperfusion in rats.

    Science.gov (United States)

    Kim, Seok-Joo; Eum, Hyun-Ae; Billiar, Timothy R; Lee, Sun-Mee

    2013-04-01

    Hepatocellular apoptosis commonly occurs in ischemia/reperfusion (I/R) injury. The binding of tumor necrosis factor (TNF) to TNF receptor 1 (TNFR1) leads to the formation of a death-inducing signaling complex (DISC), which subsequently initiates a caspase cascade resulting in apoptosis. Heme oxygenase 1 (HO-1) confers cytoprotection against cell death in I/R injury and inhibits stress-induced apoptotic pathways in vitro. This study investigated the role of HO-1 in modulating TNF/TNFR1-mediated cell death pathways in hepatic I/R injury. Rats were pretreated with hemin, an HO-1 inducer, and zinc protoporphyrin (ZnPP), an HO-1 inhibitor, before undergoing hepatic I/R. Heme oxygenase 1 activity increased after reperfusion. Ischemia/reperfusion-induced hepatocellular apoptosis was attenuated by hemin, as determined by the caspase-3 and -8 activity assays and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling). Zinc protoporphyrin eliminated the cytoprotective effect of hemin. Hepatic TNFR1 protein expression was unchanged among the experimental groups, whereas mitochondrial TNFR1 protein increased after I/R. Ischemia/reperfusion increased the quantity of DISC components, including TRADD (TNFR1-associated death domain), FADD (Fas-associated death domain), and caspase-8, as well as the assembly of DISCs within the liver. In the mitochondrial fraction, TNFR1-associated caspase-8 was increased after I/R. These increases were attenuated by hemin; zinc protoporphyrin eliminated this effect. Our findings suggest that the cytoprotective effects of HO-1 are mediated by suppression of TNF/TNFR1-mediated apoptotic signaling, specifically by modulating apoptotic DISC formation and mitochondrial TNFR1 translocation during hepatic I/R.

  2. Contribution of NMDA receptor-mediated component to the EPSP in mouse Schaffer collateral synapses under single pulse stimulation protocol.

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    Neagu, Bogdan; Strominger, Norman L; Carpenter, David O

    2008-11-13

    The degree to which NMDA receptors contribute to hippocampal CA(1) stratum radiatum excitatory postsynaptic potentials (EPSP) is a matter of debate. This experiment was designed to resolve the issue by documenting and positively identifying the elements of the NMDA dependent component in the extracellularly recorded stratum radiatum CA(1) field potential under low stimulation conditions and in the presence of physiologic levels of Mg(2+). We show that EPSP generation consists of activation of both AMPA and NMDA receptor channels, which mediate distinct components of the recorded field potential. We propose that the EPSP is a combination of two waves rather than one, which sometimes has been attributed to the exclusive activation of AMPA channels. Our data suggest that the three recorded peaks signify different events. The first peak reflects the presynaptic volley while the other two represent the actual EPSP. The first peak of the EPSP is determined mainly by flow of ions through AMPA channels. The second peak most likely is determined by the concurrence of two phenomena: ionic flow through NMDA channels and the source corresponding to the sink generated at the cell bodies in the pyramidal layer. The NMDA dependent component was recorded when Mg(2+) was present in physiological concentrations. The presynaptic volley and second peak do not saturate over a 10-fold increase of the stimulation charge and their amplitudes are highly correlated. The first peak amplitude rapidly saturates. The sensitivity of the recorded signals is different, the first peak being the most sensitive (1.25-0.26 mV/nC). Isolation of NMDA dependent components under physiological conditions when using a single pulse low stimulation protocol would allow more precise investigations of the NMDA dependent forms of synaptic plasticity.

  3. Chronic exposure to bisphenol a impairs progesterone receptor-mediated signaling in the uterus during early pregnancy

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    Li, Quanxi; Davila, Juanmahel; Bagchi, Milan K.; Bagchi, Indrani C.

    2016-01-01

    Environmental and occupational exposure to endocrine disrupting chemicals (EDCs) is a major threat to female reproductive health. Bisphenol A (BPA), an environmental toxicant that is commonly found in polycarbonate plastics and epoxy resins, has received much attention due to its estrogenic activity and high risk of chronic exposure in human. Whereas BPA has been linked to infertility and recurrent miscarriage in women, the impact of its exposure on uterine function during early pregnancy remains unclear. In a recent publication in Endocrinology, we demonstrated that prolonged exposure to an environmental relevant dose of BPA disrupts progesterone receptor-regulated uterine functions, thus affecting uterine receptivity for embryo implantation and decidua morphogenesis, two critical events for establishment and maintenance of early pregnancy. In particular we reported a marked impairment of progesterone receptor (PGR) expression and its downstream effector HAND2 in the uterine stromal cells in response to chronic BPA exposure. In an earlier study we have shown that HAND2 controls embryo implantation by repressing fibroblast growth factor (FGF) expression and the MAP kinase signaling pathway, thus inhibiting epithelial proliferation. Interestingly we observed that downregulation of PGR and HAND2 expression in uterine stroma upon BPA exposure was associated with an enhanced activation of FGFR and MAPK signaling, aberrant proliferation, and lack of uterine receptivity in the epithelium. In addition, the proliferation and differentiation of endometrial stromal cells to decidual cells, an event critical for the maintenance of early pregnancy, was severely compromised in response to BPA. This research highlight will provide an overview of our findings and discuss the potential mechanisms by which chronic BPA impairs PGR-HAND2 pathway and adversely affects implantation and the establishment of pregnancy.

  4. Degradation of Epidermal Growth Factor Receptor Mediates Dasatinib-Induced Apoptosis in Head and Neck Squamous Cell Carcinoma Cells

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    Yu-Chin Lin

    2012-06-01

    Full Text Available Epidermal growth factor receptor (EGFR is an important oncoprotein that promotes cell growth and proliferation. Dasatinib, a bcr-abl inhibitor, has been approved clinically for the treatment of chronic myeloid leukemia and demonstrated to be effective against solid tumors in vitro through Src inhibition. Here, we disclose that EGFR degradation mediated dasatinib-induced apoptosis in head and neck squamous cell carcinoma (HNSCC cells. HNSCC cells, including Ca9-22, FaDu, HSC3, SAS, SCC-25, and UMSCC1, were treated with dasatinib, and cell viability, apoptosis, and underlying signal transduction were evaluated. Dasatinib exhibited differential sensitivities against HNSCC cells. Growth inhibition and apoptosis were correlated with its inhibition on Akt, Erk, and Bcl-2, irrespective of Src inhibition. Accordingly, we found that down-regulation of EGFR was a determinant of dasatinib sensitivity. Lysosome inhibitor reversed dasatinib-induced EGFR down-regulation, and c-cbl activity was increased by dasatinib, indicating that dasatinib-induced EGFR down-regulation might be through c-cbl-mediated lysosome degradation. Increased EGFR activation by ligand administration rescued cells from dasatinib-induced apoptosis, whereas inhibition of EGFR enhanced its apoptotic effect. Estrogen receptor α (ERα was demonstrated to play a role in Bcl-2 expression, and dasatinib inhibited ERα at the pretranslational level. ERα was associated with EGFR in dasatinib-treated HNSCC cells. Furthermore, the xenograft model showed that dasatinib inhibited HSC3 tumor growth through in vivo down-regulation of EGFR and ERα. In conclusion, degradation of EGFR is a novel mechanism responsible for dasatinib-induced apoptosis in HNSCC cells.

  5. P2X7 receptor-mediated purinergic signaling promotes liver injury in acetaminophen hepatotoxicity in mice.

    Science.gov (United States)

    Hoque, Rafaz; Sohail, Muhammed Adnan; Salhanick, Steven; Malik, Ahsan F; Ghani, Ayaz; Robson, Simon C; Mehal, Wajahat Z

    2012-05-15

    Inflammation contributes to liver injury in acetaminophen (APAP) hepatotoxicity in mice and is triggered by stimulation of immune cells. The purinergic receptor P2X7 is upstream of the nod-like receptor family, pryin domain containing-3 (NLRP3) inflammasome in immune cells and is activated by ATP and NAD that serve as damage-associated molecular patterns. APAP hepatotoxicity was assessed in mice genetically deficient in P2X7, the key inflammatory receptor for nucleotides (P2X7-/-), and in wild-type mice. P2X7-/- mice had significantly decreased APAP-induced liver necrosis. In addition, APAP-poisoned mice were treated with the specific P2X7 antagonist A438079 or etheno-NAD, a competitive antagonist of NAD. Pre- or posttreatment with A438079 significantly decreased APAP-induced necrosis and hemorrhage in APAP liver injury in wild-type but not P2X7-/- mice. Pretreatment with etheno-NAD also significantly decreased APAP-induced necrosis and hemorrhage in APAP liver injury. In addition, APAP toxicity in mice lacking the plasma membrane ecto-NTPDase CD39 (CD39-/-) that metabolizes ATP was examined in parallel with the use of soluble apyrase to deplete extracellular ATP in wild-type mice. CD39-/- mice had increased APAP-induced hemorrhage and mortality, whereas apyrase also decreased APAP-induced mortality. Kupffer cells were treated with extracellular ATP to assess P2X7-dependent inflammasome activation. P2X7 was required for ATP-stimulated IL-1β release. In conclusion, P2X7 and exposure to the ligands ATP and NAD are required for manifestations of APAP-induced hepatotoxicity.

  6. Adenosine A2B receptor-mediated leukemia inhibitory factor release from astrocytes protects cortical neurons against excitotoxicity

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    Moidunny Shamsudheen

    2012-08-01

    Full Text Available Abstract Background Neuroprotective and neurotrophic properties of leukemia inhibitory factor (LIF have been widely reported. In the central nervous system (CNS, astrocytes are the major source for LIF, expression of which is enhanced following disturbances leading to neuronal damage. How astrocytic LIF expression is regulated, however, has remained an unanswered question. Since neuronal stress is associated with production of extracellular adenosine, we investigated whether LIF expression in astrocytes was mediated through adenosine receptor signaling. Methods Mouse cortical neuronal and astrocyte cultures from wild-type and adenosine A2B receptor knock-out animals, as well as adenosine receptor agonists/antagonists and various enzymatic inhibitors, were used to study LIF expression and release in astrocytes. When needed, a one-way analysis of variance (ANOVA followed by Bonferroni post-hoc test was used for statistical analysis. Results We show here that glutamate-stressed cortical neurons induce LIF expression through activation of adenosine A2B receptor subtype in cultured astrocytes and require signaling of protein kinase C (PKC, mitogen-activated protein kinases (MAPKs: p38 and ERK1/2, and the nuclear transcription factor (NF-κB. Moreover, LIF concentration in the supernatant in response to 5′-N-ethylcarboxamide (NECA stimulation was directly correlated to de novo protein synthesis, suggesting that LIF release did not occur through a regulated release pathway. Immunocytochemistry experiments show that LIF-containing vesicles co-localize with clathrin and Rab11, but not with pHogrin, Chromogranin (CgA and CgB, suggesting that LIF might be secreted through recycling endosomes. We further show that pre-treatment with supernatants from NECA-treated astrocytes increased survival of cultured cortical neurons against glutamate, which was absent when the supernatants were pre-treated with an anti-LIF neutralizing antibody. Conclusions

  7. A novel ecdysone receptor mediates steroid-regulated developmental events during the mid-third instar of Drosophila.

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    Benjamin F B Costantino

    2008-06-01

    Full Text Available The larval salivary gland of Drosophila melanogaster synthesizes and secretes glue glycoproteins that cement developing animals to a solid surface during metamorphosis. The steroid hormone 20-hydroxyecdysone (20E is an essential signaling molecule that modulates most of the physiological functions of the larval gland. At the end of larval development, it is known that 20E--signaling through a nuclear receptor heterodimer consisting of EcR and USP--induces the early and late puffing cascade of the polytene chromosomes and causes the exocytosis of stored glue granules into the lumen of the gland. It has also been reported that an earlier pulse of hormone induces the temporally and spatially specific transcriptional activation of the glue genes; however, the receptor responsible for triggering this response has not been characterized. Here we show that the coordinated expression of the glue genes midway through the third instar is mediated by 20E acting to induce genes of the Broad Complex (BRC through a receptor that is not an EcR/USP heterodimer. This result is novel because it demonstrates for the first time that at least some 20E-mediated, mid-larval, developmental responses are controlled by an uncharacterized receptor that does not contain an RXR-like component.

  8. H4 histamine receptors mediate cell cycle arrest in growth factor-induced murine and human hematopoietic progenitor cells.

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    Anne-France Petit-Bertron

    Full Text Available The most recently characterized H4 histamine receptor (H4R is expressed preferentially in the bone marrow, raising the question of its role during hematopoiesis. Here we show that both murine and human progenitor cell populations express this receptor subtype on transcriptional and protein levels and respond to its agonists by reduced growth factor-induced cell cycle progression that leads to decreased myeloid, erythroid and lymphoid colony formation. H4R activation prevents the induction of cell cycle genes through a cAMP/PKA-dependent pathway that is not associated with apoptosis. It is mediated specifically through H4R signaling since gene silencing or treatment with selective antagonists restores normal cell cycle progression. The arrest of growth factor-induced G1/S transition protects murine and human progenitor cells from the toxicity of the cell cycle-dependent anticancer drug Ara-C in vitro and reduces aplasia in a murine model of chemotherapy. This first evidence for functional H4R expression in hematopoietic progenitors opens new therapeutic perspectives for alleviating hematotoxic side effects of antineoplastic drugs.

  9. Muscarinic type 1 receptors mediate part of nitric oxide's vagal facilitatory effect in the isolated innervated rat right atrium.

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    Hogan, K; Markos, F

    2007-02-01

    We investigated whether vagal cardiac cholinergic facilitation by nitric oxide (NO) is mediated by cardiac muscarinic receptor subtypes in the vagally innervated rat right atrium in vitro. Experiments were carried out in the presence of atenolol (4 microM). The right vagus was stimulated at 4, 8, 16, 32 Hz; pulse duration 1 ms at 20 V for 20s; vagal postganglionic activation was achieved using nicotine (0.1, 0.3, 0.5, 1mM) and the effect on cardiac interval (ms) assessed. Pirenzepine (1 microM), a M1 antagonist, attenuated vagally induced increase in cardiac interval. L-Arginine (0.34 mM) superfused with pirenzepine failed to reverse this attenuation, however, L-arginine applied alone reversed the reduction vagal cardiac slowing. Similarly, sodium nitroprusside (10 microM) applied alone, and not together with pirenzepine, was able to reverse the attenuation of vagal effects caused by pirenzepine. Synthetic MT7 (1 nM) toxin, a selective M1 antagonist confirmed these results. M3 antagonism using para-fluorohexahydrosiladifenidol (p-F-HHSiD) (300 nM) and M4 antagonism with PD 102807 (200 nM) did not affect the vagally induced increase in cardiac interval. Nicotine induced increase in cardiac interval was not altered by pirenzepine. These results show that antagonism of M1 receptors on cardiac vagal preganglionic fibres reduces vagal efficacy which can be recovered by either a nitric oxide synthase substrate or a NO donor.

  10. Receptor-mediated gene delivery using polyethylenimine (PEI)coupled with polypeptides targeting FGF receptors on cells surface

    Institute of Scientific and Technical Information of China (English)

    LI Da; WANG Qing-qing; TANG Gu-ping; HUANG Hong-liang; SHEN Fen-ping; LI Jing-zhong; YU Hai

    2006-01-01

    Objective: To construct a novel kind ofnonviral gene delivery vector based on polyethylenimine (PEI) conjugated with polypeptides derived from ligand FGF with high transfection efficiency and according to tumor targeting ability. Methods:The synthetic polypeptides CR16 for binding FGF receptors was conjugated to PEI and the characters of the polypeptides including DNA condensing and particle size were determined. Enhanced efficiency and the targeting specificity of the synthesized vector were investigated in vitro and in vivo. Results: The polypeptides were successfully coupled to PEI. The new vectors PEI-CR16 could efficiently condense pDNA into particles with around 200 nm diameter. The PEI-CR16/pDNA polyplexes showed significantly greater transgene activity than PEI/pDNA in FGF receptors positive tumor cells in vitro and in vivo gene transfer, while no difference was observed in FGF receptors negative tumor cells. The enhanced transfection efficiency of PEI-CR16 could be blocked by excess free polypeptides. Conclusion: The synthesized vector could improve the efficiency of gene transfer and targeting specificity in FGF receptors positive cells. The vector had good prospect for use in cancer gene therapy.

  11. β Common Receptor Mediates Erythropoietin-Conferred Protection on OxLDL-Induced Lipid Accumulation and Inflammation in Macrophages

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    Tzong-Shyuan Lee

    2015-01-01

    Full Text Available Erythropoietin (EPO, the key factor for erythropoiesis, also protects macrophage foam cells from lipid accumulation, yet the definitive mechanisms are not fully understood. β common receptor (βCR plays a crucial role in the nonhematopoietic effects of EPO. In the current study, we investigated the role of βCR in EPO-mediated protection in macrophages against oxidized low-density lipoprotein- (oxLDL- induced deregulation of lipid metabolism and inflammation. Here, we show that βCR expression was mainly in foamy macrophages of atherosclerotic aortas from apolipoprotein E-deficient mice. Results of confocal microscopy and immunoprecipitation analyses revealed that βCR was colocalized and interacted with EPO receptor (EPOR in macrophages. Inhibition of βCR activation by neutralizing antibody or small interfering RNA (siRNA abolished the EPO-conferred protection in oxLDL-induced lipid accumulation. Furthermore, EPO-promoted cholesterol efflux and upregulation of ATP-binding cassette (ABC transporters ABCA1 and ABCG1 were prevented by pretreatment with βCR neutralizing antibody or βCR siRNA. Additionally, blockage of βCR abrogated the EPO-conferred anti-inflammatory action on oxLDL-induced production of macrophage inflammatory protein-2. Collectively, our findings suggest that βCR may play an important role in the beneficial effects of EPO against oxLDL-elicited dysfunction of macrophage foam cells.

  12. Identification and molecular characterization of cellular factors required for glucocorticoid receptor-mediated mRNA decay

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    Park, Ok Hyun; Park, Joori; Yu, Mira; An, Hyoung-Tae; Ko, Jesang; Kim, Yoon Ki

    2016-01-01

    Glucocorticoid (GC) receptor (GR) has been shown recently to bind a subset of mRNAs and elicit rapid mRNA degradation. However, the molecular details of GR-mediated mRNA decay (GMD) remain unclear. Here, we demonstrate that GMD triggers rapid degradation of target mRNAs in a translation-independent and exon junction complex-independent manner, confirming that GMD is mechanistically distinct from nonsense-mediated mRNA decay (NMD). Efficient GMD requires PNRC2 (proline-rich nuclear receptor coregulatory protein 2) binding, helicase ability, and ATM-mediated phosphorylation of UPF1 (upstream frameshift 1). We also identify two GMD-specific factors: an RNA-binding protein, YBX1 (Y-box-binding protein 1), and an endoribonuclease, HRSP12 (heat-responsive protein 12). In particular, using HRSP12 variants, which are known to disrupt trimerization of HRSP12, we show that HRSP12 plays an essential role in the formation of a functionally active GMD complex. Moreover, we determine the hierarchical recruitment of GMD factors to target mRNAs. Finally, our genome-wide analysis shows that GMD targets a variety of transcripts, implicating roles in a wide range of cellular processes, including immune responses.

  13. Presynaptic Adenosine Receptor-Mediated Regulation of Diverse Thalamocortical Short-Term Plasticity in the Mouse Whisker Pathway

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    Ferrati, Giovanni; Martini, Francisco J.; Maravall, Miguel

    2016-01-01

    Short-term synaptic plasticity (STP) sets the sensitivity of a synapse to incoming activity and determines the temporal patterns that it best transmits. In “driver” thalamocortical (TC) synaptic populations, STP is dominated by depression during stimulation from rest. However, during ongoing stimulation, lemniscal TC connections onto layer 4 neurons in mouse barrel cortex express variable STP. Each synapse responds to input trains with a distinct pattern of depression or facilitation around its mean steady-state response. As a result, in common with other synaptic populations, lemniscal TC synapses express diverse rather than uniform dynamics, allowing for a rich representation of temporally varying stimuli. Here, we show that this STP diversity is regulated presynaptically. Presynaptic adenosine receptors of the A1R type, but not kainate receptors (KARs), modulate STP behavior. Blocking the receptors does not eliminate diversity, indicating that diversity is related to heterogeneous expression of multiple mechanisms in the pathway from presynaptic calcium influx to neurotransmitter release. PMID:26941610

  14. Alzheimer’s amyloid-β peptide disturbs P2X7 receptor-mediated circadian oscillations of intracellular calcium

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    Anna Wilkaniec

    2016-12-01

    Full Text Available Recent data indicate that Alzheimer’s disease (AD is associated with disturbances of the circadian rhythm in patients. We examined the effect of amyloid-β (Aβ peptide, the main component of the senile plaques playing a critical role in the deregulation of calcium (Ca 2+ homeostasis in AD, on the circadian oscillation of cytosolic calcium (Ca 2+ levels in vitro . The experiments we carried out in human primary skin fibroblasts. This cell line was previously shown to exhibit circadian rhythms of clock genes. Moreover, the basic clock properties of these peripheral cells closely mimic those measured physiologically and behaviorally in human and do not change during aging. In this study we showed that i cytosolic Ca 2+ oscillations depend on the activation of purinergic P2X7 receptors; and ii these oscillations are abolished in the presence of Aβ. In total, our new findings may help to deepen our understanding of the molecular mechanisms involved in AD-related circadian alterations.

  15. Novel agonists for serotonin 5-HT7 receptors reverse metabotropic glutamate receptor-mediated long-term depression in the hippocampus of wild-type and Fmr1 KO mice, a model of Fragile X Syndrome

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    Lara eCosta

    2015-03-01

    Full Text Available Serotonin 5-HT7 receptors are expressed in the hippocampus and modulate the excitability of hippocampal neurons. We have previously shown that 5-HT7 receptors modulate glutamate-mediated hippocampal synaptic transmission and long-term synaptic plasticity. In particular, we have shown that activation of 5-HT7 receptors reversed metabotropic glutamate receptor-mediated long-term depression (mGluR-LTD in wild-type (wt and in Fmr1 KO mice, a mouse model of Fragile X syndrome in which mGluR-LTD is abnormally enhanced, suggesting that 5-HT7 receptor agonists might be envisaged as a novel therapeutic strategy for Fragile X syndrome. In this perspective, we have characterized the basic in vitro pharmacokinetic properties of novel molecules with high binding affinity and selectivity for 5-HT7 receptors and we have tested their effects on synaptic plasticity using patch clamp on acute hippocampal slices.Here we show that LP-211, a high affinity selective agonist of 5-HT7 receptors, reverses mGluR-LTD in wt and Fmr1 KO mice, correcting a synaptic malfunction in the mouse model of Fragile X syndrome. Among novel putative agonists of 5-HT7 receptors, the compound BA-10 displayed improved affinity and selectivity for 5-HT7 receptors and improved in vitro pharmacokinetic properties with respect to LP-211. BA-10 significantly reversed mGluR-LTD in the CA3-CA1 synapse in wt and Fmr1KO mice, indicating that BA-10 behaved as a highly effective agonist of 5-HT7 receptors and reduced exaggerated mGluR-LTD in a mouse model of Fragile X Syndrome. On the other side, the compounds RA-7 and PM-20, respectively arising from in vivo metabolism of LP-211 and BA-10, had no effect on mGluR-LTD thus did not behave as agonists of 5-HT7 receptors in our conditions.The present results provide information about the structure-activity relationship of novel 5-HT7 receptor agonists and indicate that LP-211 and BA-10 might be used as novel pharmacological tools for the therapy of

  16. Novel agonists for serotonin 5-HT7 receptors reverse metabotropic glutamate receptor-mediated long-term depression in the hippocampus of wild-type and Fmr1 KO mice, a model of Fragile X Syndrome.

    Science.gov (United States)

    Costa, Lara; Sardone, Lara M; Lacivita, Enza; Leopoldo, Marcello; Ciranna, Lucia

    2015-01-01

    Serotonin 5-HT7 receptors are expressed in the hippocampus and modulate the excitability of hippocampal neurons. We have previously shown that 5-HT7 receptors modulate glutamate-mediated hippocampal synaptic transmission and long-term synaptic plasticity. In particular, we have shown that activation of 5-HT7 receptors reversed metabotropic glutamate receptor-mediated long-term depression (mGluR-LTD) in wild-type (wt) and in Fmr1 KO mice, a mouse model of Fragile X Syndrome in which mGluR-LTD is abnormally enhanced, suggesting that 5-HT7 receptor agonists might be envisaged as a novel therapeutic strategy for Fragile X Syndrome. In this perspective, we have characterized the basic in vitro pharmacokinetic properties of novel molecules with high binding affinity and selectivity for 5-HT7 receptors and we have tested their effects on synaptic plasticity using patch clamp on acute hippocampal slices. Here we show that LP-211, a high affinity selective agonist of 5-HT7 receptors, reverses mGluR-LTD in wt and Fmr1 KO mice, correcting a synaptic malfunction in the mouse model of Fragile X Syndrome. Among novel putative agonists of 5-HT7 receptors, the compound BA-10 displayed improved affinity and selectivity for 5-HT7 receptors and improved in vitro pharmacokinetic properties with respect to LP-211. BA-10 significantly reversed mGluR-LTD in the CA3-CA1 synapse in wt and Fmr1KO mice, indicating that BA-10 behaved as a highly effective agonist of 5-HT7 receptors and reduced exaggerated mGluR-LTD in a mouse model of Fragile X Syndrome. On the other side, the compounds RA-7 and PM-20, respectively arising from in vivo metabolism of LP-211 and BA-10, had no effect on mGluR-LTD thus did not behave as agonists of 5-HT7 receptors in our conditions. The present results provide information about the structure-activity relationship of novel 5-HT7 receptor agonists and indicate that LP-211 and BA-10 might be used as novel pharmacological tools for the therapy of Fragile X Syndrome.

  17. Adenosine A1 receptor-mediated inhibition of in vitro prolactin secretion from the rat anterior pituitary

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    D.L.W. Picanço-Diniz

    2006-11-01

    Full Text Available In previous studies, we demonstrated biphasic purinergic effects on prolactin (PRL secretion stimulated by an adenosine A2 agonist. In the present study, we investigated the role of the activation of adenosine A1 receptors by (R-N6-(2-phenylisopropyladenosine (R-PIA at the pituitary level in in vitro PRL secretion. Hemipituitaries (one per cuvette in five replicates from adult male rats were incubated. Administration of R-PIA (0.001, 0.01, 0.1, 1, and 10 µM induced a reduction of PRL secretion into the medium in a U-shaped dose-response curve. The maximal reduction was obtained with 0.1 µM R-PIA (mean ± SEM, 36.01 ± 5.53 ng/mg tissue weight (t.w. treatment compared to control (264.56 ± 15.46 ng/mg t.w.. R-PIA inhibition (0.01 µM = 141.97 ± 15.79 vs control = 244.77 ± 13.79 ng/mg t.w. of PRL release was blocked by 1 µM cyclopentyltheophylline, a specific A1 receptor antagonist (1 µM = 212.360 ± 26.560 ng/mg t.w., whereas cyclopentyltheophylline alone (0.01, 0.1, 1 µM had no effect. R-PIA (0.001, 0.01, 0.1, 1 µM produced inhibition of PRL secretion stimulated by both phospholipase C (0.5 IU/mL; 977.44 ± 76.17 ng/mg t.w. and dibutyryl cAMP (1 mM; 415.93 ± 37.66 ng/mg t.w. with nadir established at the dose of 0.1 µM (225.55 ± 71.42 and 201.9 ± 19.08 ng/mg t.w., respectively. Similarly, R-PIA (0.01 µM decreased (242.00 ± 24.00 ng/mg t.w. the PRL secretion stimulated by cholera toxin (0.5 mg/mL; 1050.00 ± 70.00 ng/mg t.w.. In contrast, R-PIA had no effect (468.00 ± 34.00 ng/mg t.w. on PRL secretion stimulation by pertussis toxin (0.5 mg/mL; 430.00 ± 26.00 ng/mg t.w.. These results suggest that inhibition of PRL secretion after A1 receptor activation by R-PIA is mediated by a Gi protein-dependent mechanism.

  18. Estrogen receptor-mediated neuroprotection: The role of the Alzheimer’s disease-related gene seladin-1

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    Alessandro Peri

    2008-09-01

    Full Text Available Alessandro Peri, Mario SerioDepartment of Clinical Physiopathology, Endocrine Unit, Center for Research, Transfer and High Education on Chronic, Inflammatory, Degenerative and Neoplastic Disorders for the Development of Novel Therapies (DENOThe, University of Florence, Florence, ItalyAbstract: Experimental evidence supports a protective role of estrogen in the brain. According to the fact that Alzheimer’s disease (AD is more common in postmenopausal women, estrogen treatment has been proposed. However, there is no general consensus on the beneficial effect of estrogen or selective estrogen receptor modulators in preventing or treating AD. It has to be said that several factors may markedly affect the efficacy of the treatment. A few years ago, the seladin-1 gene (for selective Alzheimer’s disease indicator-1 has been isolated and found to be down-regulated in brain regions affected by AD. Seladin-1 has been found to be identical to the gene encoding the enzyme 3-beta-hydroxysterol delta-24-reductase, involved in the cholesterol biosynthetic pathway, which confers protection against β-amyloid-mediated toxicity and from oxidative stress, and is an effective inhibitor of caspase-3 activity, a key mediator of apoptosis. Interestingly, we found earlier that the expression of this gene is up-regulated by estrogen. Furthermore, our very recent data support the hypothesis that seladin-1 is a mediator of the neuroprotective effects of estrogen. This review will summarize the current knowledge regarding the neuroprotective effects of seladin-1 and the relationship between this protein and estrogen.Keywords: seladin-1, DHCR24, estrogen, brain, Alzheimer’s disease

  19. Smoking particles enhance endothelin A and endothelin B receptor-mediated contractions by enhancing translation in rat bronchi

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    Vikman Petter

    2006-03-01

    Full Text Available Abstract Background Smoking is known to cause chronic inflammatory changes in the bronchi and to contribute to airway hyper-reactivity, such as in bronchial asthma. To study the effect of smoking on the endothelin system in rat airways, bronchial segments were exposed to DMSO-soluble smoking particles (DSP from cigarette smoke, to nicotine and to DMSO, respectively. Methods Isolated rat bronchial segments were cultured for 24 hours in the presence or absence of DSP, nicotine or DMSO alone. Contractile responses to sarafotoxin 6c (a selective agonist for ETB receptors and endothelin-1 (an ETA and ETB receptor agonist were studied by use of a sensitive myograph. Before ET-1 was introduced, the ETB receptors were desensitized by use of S6c. The remaining contractility observed was considered to be the result of selective activation of the ETA receptors. ETA and ETB receptor mRNA expression was analyzed using real-time quantitative PCR. The location and concentration of ETA and ETB receptors were studied by means of immunohistochemistry together with confocal microscopy after overnight incubation with selective antibodies. Results After being cultured together with DSP for 24 hours the bronchial segments showed an increased contractility mediated by ETA and ETB receptors, whereas culturing them together with nicotine did not affect their contractility. The up-regulation of their contractility was blunted by cycloheximide treatment, a translational inhibitor. No significant change in the expression of ETA and ETB receptor mRNA through exposure to DMSO or to nicotine exposure alone occurred, although immunohistochemistry revealed a clear increase in ETA and ETB receptors in the smooth muscle after incubation in the presence of DSP. Taken as a whole, this is seen as the presence of a translation mechanism. Conclusion The increased contractility of rat bronchi when exposed to DSP appears to be due to a translation mechanism.

  20. Lymphotropic Virions Affect Chemokine Receptor-Mediated Neural Signaling and Apoptosis: Implications for Human Immunodeficiency Virus Type 1-Associated Dementia

    Science.gov (United States)

    Zheng, Jialin; Ghorpade, Anuja; Niemann, Douglas; Cotter, Robin L.; Thylin, Michael R.; Epstein, Leon; Swartz, Jennifer M.; Shepard, Robin B.; Liu, Xiaojuan; Nukuna, Adeline; Gendelman, Howard E.

    1999-01-01

    Chemokine receptors pivotal for human immunodeficiency virus type 1 (HIV-1) infection in lymphocytes and macrophages (CCR3, CCR5, and CXCR4) are expressed on neural cells (microglia, astrocytes, and/or neurons). It is these cells which are damaged during progressive HIV-1 infection of the central nervous system. We theorize that viral coreceptors could effect neural cell damage during HIV-1-associated dementia (HAD) without simultaneously affecting viral replication. To these ends, we studied the ability of diverse viral strains to affect intracellular signaling and apoptosis of neurons, astrocytes, and monocyte-derived macrophages. Inhibition of cyclic AMP, activation of inositol 1,4,5-trisphosphate, and apoptosis were induced by diverse HIV-1 strains, principally in neurons. Virions from T-cell-tropic (T-tropic) strains (MN, IIIB, and Lai) produced the most significant alterations in signaling of neurons and astrocytes. The HIV-1 envelope glycoprotein, gp120, induced markedly less neural damage than purified virions. Macrophage-tropic (M-tropic) strains (ADA, JR-FL, Bal, MS-CSF, and DJV) produced the least neural damage, while 89.6, a dual-tropic HIV-1 strain, elicited intermediate neural cell damage. All T-tropic strain-mediated neuronal impairments were blocked by the CXCR4 antibody, 12G5. In contrast, the M-tropic strains were only partially blocked by 12G5. CXCR4-mediated neuronal apoptosis was confirmed in pure populations of rat cerebellar granule neurons and was blocked by HA1004, an inhibitor of calcium/calmodulin-dependent protein kinase II, protein kinase A, and protein kinase C. Taken together, these results suggest that progeny HIV-1 virions can influence neuronal signal transduction and apoptosis. This process occurs, in part, through CXCR4 and is independent of CD4 binding. T-tropic viruses that traffic in and out of the brain during progressive HIV-1 disease may play an important role in HAD neuropathogenesis. PMID:10482576

  1. Bi-directional CB1 receptor-mediated cardiovascular effects of cannabinoids in anaesthetized rats: role of the paraventricular nucleus.

    Science.gov (United States)

    Grzeda, E; Schlicker, E; Luczaj, W; Harasim, E; Baranowska-Kuczko, M; Malinowska, B

    2015-06-01

    The activation of cannabinoid CB1 receptors decreases and increases blood pressure (BP) in anaesthetized and conscious rats, respectively. The aim of our study was to check the possible involvement of CB1 receptors in the paraventricular nucleus of the hypothalamus (PVN) in the cardiovascular effects of cannabinoids in rats. Methanandamide (metabolically stable analogue of the endocannabinoid anandamide) and the synthetic cannabinoid receptor agonist CP55940 were microinjected into the PVN of urethane-anaesthetized rats twice (S1 and S2, 20 min apart). Receptor antagonists were administered intravenously (i.v.) 5 min before S1. Methanandamide and CP55940 decreased blood pressure by 15 - 20%. The CB1 receptor antagonist AM251 reversed the depressor effect into a pressor response of 20 - 30%. The pressor effect of CP55940 observed in the presence of AM251 i.v. was reduced by AM251 given additionally into the PVN but not by the i.v. injection of the CB2 antagonist SR144528 or the vanilloid TRPV1 antagonist ruthenium red. In the presence of the peripherally restricted CB1 receptor antagonist AM6545, CP55940 given into the PVN increased BP by 40%. AM6545 reversed the decrease in BP induced by CP55940 i.v. into a marked increase. Bilateral chemical lesion of the PVN by kainic acid abolished all cardiovascular effects of CP55940 i.v. In conclusion, the cannabinoid CP55940 administered to the PVN of urethane-anaesthetized rats can induce depressor and pressor effects. The direction of the response probably depends on the sympathetic tone. The centrally induced hypertensive response of CP55940 can, in addition, be masked by peripheral CB1 receptors.

  2. Postsynaptic action of brain-derived neurotrophic factor attenuates alpha7 nicotinic acetylcholine receptor-mediated responses in hippocampal interneurons.

    Science.gov (United States)

    Fernandes, Catarina C; Pinto-Duarte, António; Ribeiro, Joaquim Alexandre; Sebastião, Ana M

    2008-05-21

    Nicotinic mechanisms acting on the hippocampus influence attention, learning, and memory and constitute a significant therapeutic target for many neurodegenerative, neurological, and psychiatric disorders. Here, we report that brain-derived neurotrophic factor (BDNF) (1-100 ng/ml), a member of the neurotrophin gene family, rapidly decreases alpha7 nicotinic acetylcholine receptor responses in interneurons of the hippocampal CA1 stratum radiatum. Such effect is dependent on the activation of the TrkB receptor and involves the actin cytoskeleton; noteworthy, it is compromised when the extracellular levels of the endogenous neuromodulator adenosine are reduced with adenosine deaminase (1 U/ml) or when adenosine A(2A) receptors are blocked with SCH 58261 (2-(2-furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine) (100 nm). The intracellular application of U73122 (1-[6[[(17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione) (5 mum), a broad-spectrum inhibitor of phospholipase C, or GF 109203X (bisindolylmaleimide I) (2 mum), a general inhibitor of protein kinase C isoforms, blocks BDNF-induced inhibition of alpha7 nicotinic acetylcholine receptor function. Moreover, in conditions of simultaneous intracellular dialysis of the fast Ca(2+) chelator BAPTA (10 mm) and removal of extracellular Ca(2+) ions, the inhibitory action of BDNF is further prevented. The present findings disclose a novel target for rapid actions of BDNF that might play important roles on synaptic transmission and plasticity in the brain.

  3. L-5-HTP facilitates the electrically stimulated flexor reflex in pithed rats: evidence for 5-HT2-receptor mediation.

    Science.gov (United States)

    Skarsfeldt, T; Arnt, J; Hyttel, J

    1990-02-06

    Different serotonin (5-HT) receptor agonists were tested on the electrically stimulated flexor reflex in pithed rats. The 5-HT2 receptor agonist, (1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane) [+/-)DOI), the mixed 5-HT1/5-HT2 receptor agonist, quipazine, and the 5-HT precursor, l-5-HTP, showed agonistic activity upon intravenous injection while 5-HT was without effect. A combination of the peripheral decarboxylase inhibitor, Ro 4-4602 (benzerazide), the specific 5-HT-uptake inhibitor, citalopram, and l-5-HTP induced a prolonged (greater than 3 h) increase of the flexor reflex in pithed rats. Different compounds were tested for an inhibitory effect against this l-5-HTP-induced flexor reflex. The 5-HT2 antagonists (ketanserin, methergoline and methiothepin) were potent antagonists. (-)Alprenolol (5-HT1A and 5-HT1B receptor antagonist) and the 5-HT3-receptor antagonist, ICS 205-930, were without an antagonistic effect. The inhibitory potencies in the reflex model (l-5-HTP, citalopram and Ro 4-4602) were significantly correlated (r = 0.83, P less than 0.01, r2 = 0.69) with the potencies to inhibit l-5-HTP-induced head twitches and quipazine-induced head twitches (r = 0.81, P less than 0.01, r2 = 0.66). There was less correlation (r = 0.75, P less than 0.01, r2 = 0.56) with the affinities for 5-HT2 receptors in vitro. There was no significant correlation between inhibitory potencies in the reflex model and affinities for dopamine (DA) D-2 receptors or alpha 1-adrenoceptors (r2 = 0.13 and 0.14, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

  4. Glycine and GABAA receptors mediate tonic and phasic inhibitory processes that contribute to prepulse inhibition in the goldfish startle network

    Directory of Open Access Journals (Sweden)

    Paul C.P. Curtin

    2015-03-01

    Full Text Available Prepulse inhibition (PPI is understood as an inhibitory process that attenuates sensory flow during early stages (20-1000ms of information processing. Here, we applied in vivo electrophysiology and pharmacology to determine if prepulse inhibition (PPI is mediated by glycine receptors (GlyRs and/or GABAA receptors (GABAARs in the goldfish auditory startle circuit. Specifically, we used selective antagonists to dissect the contributions of target receptors on sound-evoked postsynaptic potentials (PSPs recorded in the neurons that initiate startle, the Mauthner-cells (M-cell. We found that strychnine, a GlyR antagonist, disrupted a fast-activated (5 ms and rapidly (< 50ms decaying (feed-forward inhibitory process that disrupts PPI at 20 ms prepulse/pulse inter-stimulus intervals (ISI. Additionally we observed increases of the evoked postsynaptic potential (PSP peak amplitude (+87.43 ± 21.53%; N=9 and duration (+204 ± 48.91%, N=9. In contrast, treatment with bicuculline, a GABAAR antagonist, caused a general reduction in PPI across all tested ISIs (20-500 ms, essentially eliminating PPI at ISIs from 20-100 ms. Bicuculline also increased PSP peak amplitude (+133.8 ± 10.3%, N=5 and PSP duration (+284.95 ± 65.64%, N=5. Treatment with either antagonist also tonically increased post-synaptic excitability in the M-cells, reflected by an increase in the magnitude of antidromically-evoked action potentials (APs by 15.07 ± 3.21%, N=7 and 16.23 ± 7.08%, N=5 for strychnine and bicuculline, respectively. These results suggest that GABAARs and GlyRs are functionally segregated to short- and longer-lasting sound-evoked (phasic inhibitory processes that contribute to PPI, with the mediation of tonic inhibition by both receptor systems being critical for gain control within the M-cell startle circuit.

  5. Ventral midbrain NTS1 receptors mediate conditioned reward induced by the neurotensin analogue, D-Tyr[11]neurotensin

    Directory of Open Access Journals (Sweden)

    Khalil eRouibi

    2015-12-01

    Full Text Available The present study was aimed at characterizing the mechanisms by which neurotensin (NT is acting within the ventral midbrain to induce a psychostimulant-like effect. In a first experiment, we determine which subtype(s of NT receptors is involved in the reward-inducing effect of ventral midbrain microinjection of NT using the conditioned place-preference (CPP paradigm. In a second study, we used in vitro patch clamp recording technique to characterize the NT receptor subtype(s involved in the modulation of glutamatergic neurotransmission (excitatory post-synaptic current, EPSC in ventral tegmental neurons that expressed (Ih+, or do not express (Ih-, a hyperpolarization-activated cationic current. Behavioral studies were performed with adult male Long-Evans rats while electrophysiological recordings were obtained from brain slices of rat pups aged between 14 and 21 days. Results show that bilateral ventral midbrain microinjections of 1.5 and 3 nmol of D-Tyr[11]NT induced a CPP that was respectively attenuated or blocked by co-injection with 1.2 nmol of the NTS1/NTS2 antagonist, SR142948, and the preferred NTS1 antagonist, SR48692. In electrophysiological experiments, D-Tyr[11]NT (0.01-0.5 M attenuated glutamatergic EPSC in Ih+ but enhanced it in Ih- neurons. The attenuation effect (Ih+ neurons was blocked by SR142948 (0.1 M while the enhancement effect (Ih- neurons was blocked by both antagonists (0.1 M. These findings suggest that i NT is acting on ventral midbrain NTS1 receptors to induce a rewarding effect and ii that this psychostimulant-like effect could be due to a direct action of NT on dopamine neurons and/or an enhancement of glutamatergic inputs to non-dopamine (Ih- neurons.

  6. Increase in neurokinin-1 receptor-mediated colonic motor response in a rat model of irritable bowel syndrome

    Institute of Scientific and Technical Information of China (English)

    Jun-Ho La; Tae-Wan Kim; Tae-Sik Sung; Hyn-Ju Kim; Jeom-Yong Kim; Il-Suk Yang

    2005-01-01

    AIM: Irritable bowel syndrome (IBS) is a functional bowel disorder. Its major symptom is bowel dysmotility, yet the mechanism of the symptom is poorly understood. Since the neurokinin-1 receptor (NK1R)-mediated signaling in the gut is important in the control of normal bowel motor function,we aimed to investigate whether the NK1R-mediated bowel motor function was altered in IBS, using a rat IBS model that was previously reported to show colonic dysmotility in response to restraint stress.METHODS: IBS symptoms were produced in male SpragueDawley rats by inducing colitis with acetic acid. Rats were left to recover from colitis for 6 d, and used for experiments 7 d post-induction of colitis. Motor activities of distal colon were recorded in vitro.RESULTS: The contractile sensitivity of isolated colon to a NK1R agonist [Sar9, Met(O2)11]-substance P (1-30 nmol/L)was higher in IBS rats than that in normal rats. After the enteric neurotransmission was blocked by tetrodotoxin (TTX, 1 μmol/L), the contractile sensitivity to the NK1R agonist was increased in normal colon but not in IBS rat colon. The NK1R agonist-induced contraction was not different between the two groups when the agonist was challenged to the TTX-treated colon or the isolated colonic myocytes. A nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME, 100 μmol/L) augmented the NK1R agonist-induced contraction only in normal rat colon.CONCLUSION: These results suggest that the NK1R-meidated colonic motor response is increased in IBS rats, due to the decrease in the nitrergic inhibitory neural component.

  7. Rostral Ventrolateral Medulla EP3 Receptor Mediates the Sympathoexcitatory and Pressor Effects of Prostaglandin E2 in Conscious Rats.

    Science.gov (United States)

    Rezq, Samar; Abdel-Rahman, Abdel A

    2016-11-01

    Whereas few studies have dealt with the central sympathoexcitatory action of the inflammatory prostanoid prostaglandin E2 (PGE2), there is no information on the expression and cardiovascular function of different PGE2 (EP) receptors in one of the major cardiovascular-regulating nuclei, the rostral ventrolateral medulla (RVLM). The current study aimed at filling this knowledge gap as well as elucidating the implicated molecular mechanisms. To achieve these goals, we showed the expression of EP2, EP3, and EP4 receptors in the RVLM and investigated their cardiovascular roles in conscious rats, ex vivo as well as in cultured PC12 cells. Intra-RVLM PGE2 significantly increased blood pressure and sympathetic dominance (spectral analysis). Studies with selective EP receptor subtype agonists and antagonists showed that these PGE2-evoked responses were only replicated by intra-RVLM activation of the EP3 receptor with its agonist sulprostone. The RVLM of PGE2-treated rats exhibited increases in c-Fos expression and extracellular signal-regulated kinase 1/2 and neuronal nitric oxide synthase phosphorylation along with oxidative stress, and PGE2 increased l-glutamate release in PC12 cells (surrogates of RVLM neurons). Abrogation of the PGE2-evoked pressor and biochemical responses only occurred following EP3 receptor blockade (N-[(5-Bromo-2-methoxyphenyl)sulfonyl]-3-[2-(2-naphthalenylmethyl)phenyl]-2-propenamide, L-798106). These findings suggest the dependence of RVLM PGE2-mediated sympathoexcitation/pressor response on local EP3 receptor signaling in conscious rats, and highlight central EP3 receptor blockade as a potential therapeutic modality for hypertension management. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  8. EP2 and EP4 receptors mediate PGE2 induced relaxation in murine colonic circular muscle: pharmacological characterization.

    Science.gov (United States)

    Martinez-Cutillas, M; Mañé, N; Gallego, D; Jimenez, M; Martin, M T

    2014-12-01

    Prostaglandin E2 (PGE2) is a regulator of gastrointestinal motility that might be involved in impaired motor function associated to gut inflammation. The aim of the present work is to pharmacologically characterize responses to exogenous and endogenous PGE2 in the mouse colon targeting EP2 and EP4 receptors. Wild type (WT) and EP2 receptor knockout (EP2-KO) mice were used to characterize PGE2 and butaprost (EP2 receptor agonist) effects on smooth muscle resting membrane potential and myogenic contractility in circularly oriented colonic preparations. In WT animals, PGE2 and butaprost concentration-dependently inhibited spontaneous contractions and hyperpolarized smooth muscle cells. Combination of both EP2 (PF-04418948 0.1μM) and EP4 receptor antagonists (L-161,982 10μM) was needed to block both electrical and mechanical PGE2 responses. Butaprost inhibitory responses (both electrical and mechanical) were totally abolished by PF-04418948 0.1μM. In EP2-KO mice, PGE2 (but not butaprost) concentration-dependently inhibited spontaneous contractions and hyperpolarized smooth muscle cells. In EP2-KO mice, PGE2 inhibition of spontaneous contractility and hyperpolarization was fully antagonized by L-161,982 10μM. In WT animals, EP2 and EP4 receptor antagonists caused a smooth muscle depolarization and an increase in spontaneous mechanical activity. PGE2 responses in murine circular colonic layer are mediated by post-junctional EP2 and EP4 receptors. PF-04418948 and L-161,982 are selective EP2 and EP4 receptor antagonists that inhibit PGE2 responses. These antagonists might be useful pharmacological tools to limit prostaglandin effects associated to dismotility in gut inflammatory processes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Delta-subunit containing GABAA-receptors mediate tonic inhibition in paracapsular cells of the mouse amygdala

    Directory of Open Access Journals (Sweden)

    Anne eMarowsky

    2014-03-01

    Full Text Available The intercalated paracapsular cells (pcs are small GABAergic interneurons that form densely populated clusters surrounding the basolateral (BLA complex of the amygdala. Their main task in the amygdala circuitry appears to be the control of information flow, as they act as an inhibitory interface between input and output nuclei. Modulation of their activity is thus thought to affect amygdala output and the generation of fear and anxiety. Recent evidence indicates that pcs express benzodiazepine (BZ-sensitive GABAA receptor (GABAAR variants containing the α2- and α3-subunit for transmission of postsynaptic currents, yet little is known about the expression of extrasynaptic GABAARs, mediating tonic inhibition and regulating neuronal excitability. Here, we show that pcs from the lateral and medial intercalated cell cluster (l- and mITC, respectively express a tonic GABAergic conductance that could be significantly increased in a concentration-dependent manner by the δ-preferring GABAAR agonist THIP (0.5-10 µM, but not by the BZ diazepam (1 µM. The neurosteroid THDOC (300 nM also increased tonic currents in pcs significantly, but only in the presence of additional GABA (5 µM. Immunohistochemical stainings revealed that both the δ-GABAAR and the α4-GABAAR subunit are expressed throughout all ITCs, while no staining for the α5-GABAAR subunit could be detected. Moreover, 1 µM THIP dampened excitability in pcs most likely by increasing shunting inhibition. In line with this, THIP significantly decreased lITC-generated inhibition in target cells residing in the BLA nucleus by 30%. Taken together these results demonstrate for the first time that pcs express a tonic inhibitory conductance mediated most likely by α4/δ-containing GABAARs. This data also suggest that δ-GABAAR targeting compounds might possibly interfere with pcs-related neuronal processes such as fear extinction.

  10. Type I (CD64) and type II (CD32) Fc gamma receptor-mediated phagocytosis by human blood dendritic cells.

    Science.gov (United States)

    Fanger, N A; Wardwell, K; Shen, L; Tedder, T F; Guyre, P M

    1996-07-15

    Three classes of Fc receptors for IgG, Fc gamma RI (CD64), Fc gamma RII (CD32), and Fc gamma RIII (CD16), are expressed on blood leukocytes. Although Fc gamma R are important phagocytic receptors on phagocytes, most reports suggest that dendritic cells lack Fc gamma R-mediated phagocytosis and express significant levels of only CD32. We now report that phagocytically active forms of both CD64 and CD32 are expressed significantly on at least one subset of human blood dendritic cells. Countercurrent elutriation and magnetic bead selection were used to rapidly enrich subsets of blood dendritic cells (CD33brightCD14-HLA-DRbrightCD83-) and monocytes (CD33brightCD14brightHLA-DRdimCD83-). Upon culture for 2 days, dendritic cells became CD83-positive and markedly increased HLA-DR expression, whereas monocytes did not express CD83 and exhibited reduced levels of HLA-DR. Constitutive CD64 expression was identified on this circulating dendritic cell population, but at a lower level than on monocytes. CD64 expression by dendritic cells and monocytes did not decrease during 2 days in culture, and was up-regulated on both cell types following incubation with IFN-gamma. Freshly isolated blood dendritic cells performed CD64- and CD32-mediated phagocytosis, although at a lower level than monocytes. Dendritic cells generated by culture of adherent mononuclear cells in granulocyte-macrophage CSF and IL-4 also up-regulated CD64 following IFN-gamma stimulation, and mediated CD64-dependent phagocytosis. These results indicate that both CD64 and CD32 expressed on blood dendritic cells may play a role in uptake of foreign particles and macromolecules through a phagocytic mechanism before trafficking to T cell-reactive areas.

  11. Enhancement of NMDA receptor-mediated excitatory postsynaptic currents by gp120-treated macrophages: implications for HIV-1-associated neuropathology.

    Science.gov (United States)

    Yang, Jianming; Hu, Dehui; Xia, Jianxun; Liu, Jianuo; Zhang, Gang; Gendelman, Howard E; Boukli, Nawal M; Xiong, Huangui

    2013-09-01

    A plethora of prior studies has linked HIV-1-infected and immune activated brain mononuclear phagocytes (MP; blood borne macrophages and microglia) to neuronal dysfunction. These are modulated by N-methyl-D-aspartate receptor (NMDAR) antagonists and supporting their relevance for HIV-1-associated nervous system disease. The role of NMDAR subsets in HIV-1-induced neuronal injury, nonetheless, is poorly understood. To this end, we investigated conditioned media from HIV-1gp120-treated human monocyte-derived-macrophages (MDM) for its abilities to affect NMDAR-mediated excitatory postsynaptic currents (EPSC(NMDAR)) in rat hippocampal slices. Bath application of gp120-treated MDM-conditioned media (MCM) produced an increase of EPSC(NMDAR). In contrast, control (untreated) MCM had limited effects on EPSC(NMDAR). Testing NR2A NMDAR (NR2AR)-mediated EPSC (EPSC(NR2AR)) and NR2B NMDAR (NR2BR)-mediated EPSC (EPSC(NR2BR)) for MCM showed significant increased EPSC(NR2BR) when compared to EPSC(NR2AR) enhancement. When synaptic NR2AR-mediated EPSC was blocked by bath application of MK801 combined with low frequency stimulations, MCM retained its ability to enhance EPSC(NMDAR) evoked by stronger stimulations. This suggested that increase in EPSC(NMDAR) was mediated, in part, through extra-synaptic NR2BR. Further analyses revealed that the soluble factors with low (NR2BR but not NR2AR blockers. Taken together, these results indicate that macrophage secretory products induce neuronal injury through extra-synaptic NR2BRs.

  12. Spinal orexin-1 receptors mediate anti-hyperalgesic effects of intrathecally-administered orexins in diabetic neuropathic pain model rats.

    Science.gov (United States)

    Kajiyama, Seiji; Kawamoto, Masashi; Shiraishi, Seiji; Gaus, Syafruddin; Matsunaga, Aki; Suyama, Hidemichi; Yuge, Osafumi

    2005-05-17

    Orexin-A and orexin-B are endogenous ligands of orexin receptors that contain orexin-1 and orexin-2. Activation of the orexinergic system can produce antinociceptive effects in acute inflammatory, mono-neuropathic, and postoperative pain animal models, though the effects of orexins on diabetic neuropathic pain have not been previously investigated. In this study, we studied the anti-hyperalgesic effects of intrathecally administered orexins in a streptozotocin-induced diabetic rat. First, dose-dependent effects were investigated by measuring hind paw withdrawal thresholds in response to noxious-heat and punctate stimuli, after which orexin levels in the cerebrospinal fluid of diabetic rats were measured and compared with those of normal rats using a radioimmunoassay method. The functional role of spinal orexin-1 receptors with the anti-hyperalgesic effects of orexins was also investigated using intrathecal pretreatment with SB-334867, a selective orexin-1 receptor antagonist. Intrathecally administered orexins produced an antinociceptive effect in diabetic rats, however, not in normal rats, though the orexin levels in the cerebrospinal fluid of diabetic rats were similar to those in normal rats. In addition, the anti-hyperalgesic effects of orexins were significantly inhibited by pretreatment with SB-334867. These findings demonstrate that the anti-hyperalgesic effects of orexins in diabetic rats are unlikely due to any direct effect by the supplement on decreased endogenous orexins in the cerebrospinal fluid and that orexin-1 receptors in the spinal cord may be involved in the modulation of nociceptive transmission in diabetic neuropathy. We conclude that the spinal orexinergic system may be a possible target for elucidating the mechanisms of diabetes-induced hyperalgesia.

  13. Ins(1,4,5)P3 receptor-mediated Ca2+ signaling and autophagy induction are interrelated.

    Science.gov (United States)

    Decuypere, Jean-Paul; Welkenhuyzen, Kirsten; Luyten, Tomas; Ponsaerts, Raf; Dewaele, Michael; Molgó, Jordi; Agostinis, Patrizia; Missiaen, Ludwig; De Smedt, Humbert; Parys, Jan B; Bultynck, Geert

    2011-12-01

    The role of intracellular Ca2+ signaling in starvation-induced autophagy remains unclear. Here, we examined Ca2+ dynamics during starvation-induced autophagy and the underlying molecular mechanisms. Tightly correlating with autophagy stimulation, we observed a remodeling of the Ca2+ signalosome. First, short periods of starvation (1 to 3 h) caused a prominent increase of the ER Ca2+-store content and enhanced agonist-induced Ca2+ release. The mechanism involved the upregulation of intralumenal ER Ca2+-binding proteins, calreticulin and Grp78/BiP, which increased the ER Ca2+-buffering capacity and reduced the ER Ca2+ leak. Second, starvation led to Ins(1,4,5)P3R sensitization. Immunoprecipitation experiments showed that during starvation Beclin 1, released from Bcl-2, first bound with increasing efficiency to Ins(1,4,5)P3Rs; after reaching a maximal binding after 3 h, binding, however, decreased again. The interaction site of Beclin 1 was determined to be present in the N-terminal Ins(1,4,5)P3-binding domain of the Ins(1,4,5)P3R. The starvation-induced Ins(1,4,5)P3R sensitization was abolished in cells treated with BECN1 siRNA, but not with ATG5 siRNA, pointing toward an essential role of Beclin 1 in this process. Moreover, recombinant Beclin 1 sensitized Ins(1,4,5)P3Rs in 45Ca2+-flux assays, indicating a direct regulation of Ins(1,4,5)P3R activity by Beclin 1. Finally, we found that Ins(1,4,5)P3R-mediated Ca2+ signaling was critical for starvation-induced autophagy stimulation, since the Ca2+ chelator BAPTA-AM as well as the Ins(1,4,5)P3R inhibitor xestospongin B abolished the increase in LC3 lipidation and GFP-LC3-puncta formation. Hence, our results indicate a tight and essential interrelation between intracellular Ca2+ signaling and autophagy stimulation as a proximal event in response to starvation.

  14. Identification of putative estrogen receptor-mediated endocrine disrupting chemicals using QSAR- and structure-based virtual screening approaches

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Liying; Sedykh, Alexander; Tripathi, Ashutosh [Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC (United States); Zhu, Hao [The Rutgers Center for Computational and Integrative Biology, Rutgers University, Camden, NJ (United States); Department of Chemistry, Rutgers University, Camden, NJ (United States); Afantitis, Antreas; Mouchlis, Varnavas D.; Melagraki, Georgia [NovaMechanics Ltd., Nicosia (Cyprus); Rusyn, Ivan, E-mail: iir@unc.edu [Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC (United States); Tropsha, Alexander, E-mail: alex_tropsha@unc.edu [Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC (United States)

    2013-10-01

    Identification of endocrine disrupting chemicals is one of the important goals of environmental chemical hazard screening. We report on the development of validated in silico predictors of chemicals likely to cause estrogen receptor (ER)-mediated endocrine disruption to facilitate their prioritization for future screening. A database of relative binding affinity of a large number of ERα and/or ERβ ligands was assembled (546 for ERα and 137 for ERβ). Both single-task learning (STL) and multi-task learning (MTL) continuous quantitative structure–activity relationship (QSAR) models were developed for predicting ligand binding affinity to ERα or ERβ. High predictive accuracy was achieved for ERα binding affinity (MTL R{sup 2} = 0.71, STL R{sup 2} = 0.73). For ERβ binding affinity, MTL models were significantly more predictive (R{sup 2} = 0.53, p < 0.05) than STL models. In addition, docking studies were performed on a set of ER agonists/antagonists (67 agonists and 39 antagonists for ERα, 48 agonists and 32 antagonists for ERβ, supplemented by putative decoys/non-binders) using the following ER structures (in complexes with respective ligands) retrieved from the Protein Data Bank: ERα agonist (PDB ID: 1L2I), ERα antagonist (PDB ID: 3DT3), ERβ agonist (PDB ID: 2NV7), and ERβ antagonist (PDB ID: 1L2J). We found that all four ER conformations discriminated their corresponding ligands from presumed non-binders. Finally, both QSAR models and ER structures were employed in parallel to virtually screen several large libraries of environmental chemicals to derive a ligand- and structure-based prioritized list of putative estrogenic compounds to be used for in vitro and in vivo experimental validation. - Highlights: • This is the largest curated dataset inclusive of ERα and β (the latter is unique). • New methodology that for the first time affords acceptable ERβ models. • A combination of QSAR and docking enables prediction of affinity and function.

  15. NR4A nuclear receptors mediate carnitine palmitoyltransferase 1A gene expression by the rexinoid HX600

    Energy Technology Data Exchange (ETDEWEB)

    Ishizawa, Michiyasu [Division of Biochemistry, Department of Biomedical Sciences, Nihon University School of Medicine, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610 (Japan); Kagechika, Hiroyuki [Graduate School of Biomedical Science, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062 (Japan); Makishima, Makoto, E-mail: makishima.makoto@nihon-u.ac.jp [Division of Biochemistry, Department of Biomedical Sciences, Nihon University School of Medicine, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610 (Japan)

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer The function of RXR heterodimers with NR4 receptors remains unknown. Black-Right-Pointing-Pointer The RXR ligand HX600 induces expression of carnitine palmitoyltransferase 1A (CPT1A). Black-Right-Pointing-Pointer HX600-induced CPT1A expression is mediated by the NR4 receptors, Nur77 and NURR1. Black-Right-Pointing-Pointer CPT1A induction by HX600 is not mediated by de novo protein synthesis. Black-Right-Pointing-Pointer CPT1A could be a target of the Nur77-RXR and NURR1-RXR heterodimers. -- Abstract: Retinoid X receptors (RXRs) are members of the nuclear receptor superfamily and can be activated by 9-cis retinoic acid (9CRA). RXRs form homodimers and heterodimers with other nuclear receptors such as the retinoic acid receptor and NR4 subfamily nuclear receptors, Nur77 and NURR1. Potential physiological roles of the Nur77-RXR and NURR1-RXR heterodimers have not been elucidated. In this study, we identified a gene regulated by these heterodimers utilizing HX600, a selective RXR agonist for Nur77-RXR and NURR1-RXR. While 9CRA induced many genes, including RAR-target genes, HX600 effectively induced only carnitine palmitoyltransferase 1A (CPT1A) in human teratocarcinoma NT2/D1 cells, which express RXR{alpha}, Nur77 and NURR1. HX600 also increased CPT1A expression in human embryonic kidney (HEK) 293 cells and hepatocyte-derived HepG2 cells. Although HX600 induced CPT1A less effectively than 9CRA, overexpression of Nur77 or NURR1 increased the HX600 response to levels similar to 9CRA in NT2/D1 and HEK293 cells. A dominant-negative form of Nur77 or NURR1 repressed the induction of CPT1A by HX600. A protein synthesis inhibitor did not alter HX600-dependent CPT1A induction. Thus, the rexinoid HX600 directly induces expression of CPT1A through a Nur77 or NURR1-mediated mechanism. CPT1A, a gene involved in fatty acid {beta}-oxidation, could be a target of RXR-NR4 receptor heterodimers.

  16. The Bcl-2 gene polymorphism rs956572AA increases inositol 1,4,5-trisphosphate receptor-mediated endoplasmic reticulum calcium release in subjects with bipolar disorder.

    Science.gov (United States)

    Machado-Vieira, Rodrigo; Pivovarova, Natalia B; Stanika, Ruslan I; Yuan, Peixiong; Wang, Yun; Zhou, Rulun; Zarate, Carlos A; Drevets, Wayne C; Brantner, Christine A; Baum, Amber; Laje, Gonzalo; McMahon, Francis J; Chen, Guang; Du, Jing; Manji, Husseini K; Andrews, S Brian

    2011-02-15

    Bipolar disorder (BPD) is characterized by altered intracellular calcium (Ca(2+)) homeostasis. Underlying mechanisms involve dysfunctions in endoplasmic reticulum (ER) and mitochondrial Ca(2+) handling, potentially mediated by B-cell lymphoma 2 (Bcl-2), a key protein that regulates Ca(2+) signaling by interacting directly with these organelles, and which has been implicated in the pathophysiology of BPD. Here, we examined the effects of the Bcl-2 gene single nucleotide polymorphism (SNP) rs956572 on intracellular Ca(2+) dynamics in patients with BPD. Live cell fluorescence imaging and electron probe microanalysis were used to measure intracellular and intra-organelle free and total calcium in lymphoblasts from 18 subjects with BPD carrying the AA, AG, or GG variants of the rs956572 SNP. Analyses were carried out under basal conditions and in the presence of agents that affect Ca(2+) dynamics. Compared with GG homozygotes, variant AA-which expresses significantly reduced Bcl-2 messenger RNA and protein-exhibited elevated basal cytosolic Ca(2+) and larger increases in inositol 1,4,5-trisphosphate receptor-mediated cytosolic Ca(2+) elevations, the latter in parallel with enhanced depletion of the ER Ca(2+) pool. The aberrant behavior of AA cells was reversed by chronic lithium treatment and mimicked in variant GG by a Bcl-2 inhibitor. In contrast, no differences between SNP variants were found in ER or mitochondrial total Ca(2+) content or in basal store-operated Ca(2+) entry. These results demonstrate that, in patients with BPD, abnormal Bcl-2 gene expression in the AA variant contributes to dysfunctional Ca(2+) homeostasis through a specific ER inositol 1,4,5-trisphosphate receptor-dependent mechanism. Copyright © 2011 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  17. Striatal adenosine A{sub 2A} receptor-mediated positron emission tomographic imaging in 6-hydroxydopamine-lesioned rats using [{sup 18}F]-MRS5425

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharjee, Abesh Kumar; Lang Lixin; Jacobson, Orit [Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892 (United States); Shinkre, Bidhan [Chemical Biology Unit, Laboratory of Cell Biochemistry and Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 (United States); Ma Ying [Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892 (United States); Niu Gang [Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892 (United States); Department of Radiology and Imaging Sciences, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892 (United States); Trenkle, William C. [Chemical Biology Unit, Laboratory of Cell Biochemistry and Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 (United States); Jacobson, Kenneth A. [Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 (United States); Chen Xiaoyuan [Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892 (United States); Kiesewetter, Dale O., E-mail: dk7k@nih.gov [Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892 (United States)

    2011-08-15

    Introduction: A{sub 2A} receptors are expressed in the basal ganglia, specifically in striatopallidal GABAergic neurons in the striatum (caudate-putamen). This brain region undergoes degeneration of presynaptic dopamine projections and depletion of dopamine in Parkinson's disease. We developed an {sup 18}F-labeled A{sub 2A} analog radiotracer ([{sup 18}F]-MRS5425) for A{sub 2A} receptor imaging using positron emission tomography (PET). We hypothesized that this tracer could image A{sub 2A} receptor changes in the rat model for Parkinson's disease, which is created following unilateral injection of the monoaminergic toxin 6-hydroxydopamine (6-OHDA) into the substantia nigra. Methods: [{sup 18}F]-MRS5425 was injected intravenously in anesthetized rats, and PET imaging data were collected. Image-derived percentage injected doses per gram (%ID/g) in regions of interest was measured in the striatum of normal rats and in rats unilaterally lesioned with 6-OHDA after intravenous administration of saline (baseline), D{sub 2} agonist quinpirole (1.0 mg/kg) or D{sub 2} antagonist raclopride (6.0 mg/kg). Results: Baseline %ID/g reached a maximum at 90 s and maintained plateau for 3.5 min, and then declined slowly thereafter. In 6-OHDA-lesioned rats, %ID/g was significantly higher in the lesioned side compared to the intact side, and the baseline total %ID/g (data from both hemispheres were combined) was significantly higher compared to quinpirole stimulation starting from 4.5 min until the end of acquisition at 30 min. Raclopride did not produce any change in uptake compared to baseline or between the hemispheres. Conclusion: Thus, increase of A{sub 2A} receptor-mediated uptake of radioactive MRS5425 could be a superior molecular target for Parkinson's imaging.

  18. Prostaglandin (PG) FP and EP1 receptors mediate PGF2alpha and PGE2 regulation of interleukin-1beta expression in Leydig cell progenitors.

    Science.gov (United States)

    Walch, Laurence; Clavarino, Emanuela; Morris, Patricia L

    2003-04-01

    Prostaglandins (PG) mediate IL-1beta regulation of several interleukin mRNAs in progenitor Leydig cells. PGE(2) and PGF(2alpha) potently reverse indomethacin (INDO; a cyclooxygenase inhibitor) inhibition of IL-1beta autoinduction. IL-1beta increases PGE(2) and PGF(2alpha) production. To determine the PG receptors involved in this regulation, this study established by RT-PCR and Western analyses which specific receptors for PGE(2) (EP receptors) and PGF(2alpha) (FP receptors) are expressed in progenitors. Pharmacological characterization of receptors involved in PGE(2) and PGF(2alpha) regulation of IL-1beta mRNA levels was ascertained using real-time PCR analyses. FP, EP(1), EP(2), and EP(4) receptor mRNAs and proteins, and an EP(3) receptor subtype were detected. IL-1beta treatment (24-h) significantly decreased EP(1) receptor levels; INDO abrogated this down-regulation. FP, EP(2), and EP(4) receptor levels increased after IL-1beta and IL-1beta + INDO. A selective FP agonist, cloprostenol (0.1 micro M), and PGF(2alpha) (10 micro M) had similar effects on IL-1beta mRNA levels in progenitors treated with IL-1beta + INDO. None of the EP(2)/EP(4) agonists [butaprost, misoprostol, or 11-deoxy PGE(1) (10 micro M)] affected IL-1beta mRNA levels. In contrast, EP(1)/EP(3) agonists (17-phenyl trinor PGE(2) and sulprostone) increased IL-1beta mRNAs in a dose-dependent manner. EP(1) receptor subtype-selective antagonist, SC-51322, blocked IL-1beta-induced and [IL-1beta + INDO + 17-phenyl trinor PGE(2)]-induced increases in IL-1beta mRNAs. Taken together, our data demonstrate that FP and EP(1) receptors mediate PGF(2alpha) and PGE(2) induction of progenitor IL-1beta expression.

  19. Enhancing the receptor-mediated cell uptake of PLGA nanoparticle for targeted drug delivery by incorporation chitosan onto the particle surface

    Science.gov (United States)

    Jiang, Guoqiang; Tang, Shifu; Chen, Xuelan; Ding, Fuxin

    2014-06-01

    Cationic polymer chitosan (CS) and target ligand were both incorporated onto nanoparticles (NPs) to enhance the cell uptake by integration of electrostatic interaction and receptor-mediated internalization. CS and biotin-contained amphipathic polymer biotin-poly(ethylene glycol)-poly(lactic acid) (biotin-PEG-PLA) were simultaneously decorated on the poly(lactic- co-glycolic acid) (PLGA) NPs surface in one step during the o/w solvent evaporation procedure. The incorporation of CS increased the zeta potential of the NPs to positive value and showed little impacts on particle size and biotin density. Cell uptake was investigated in vitro using human hepatic carcinoma cell lines SMMC-7721. The CS and biotin co-decorated NPs (CS-B-NPs) presented significantly higher cell uptake than that of the mono biotin-decorated NPs (B-NPs). In acid environment, as CS-B-NPs are more positive charged, cell uptake of CS-B-NPs is further increased, which is 3.8-fold as much as that of the undecorated NPs (U-NPs) and 1.9-fold higher than that of B-NPs at pH 6.6. When either the ligand density was reduced within limited or the particle size was slightly increased, cell uptake of CS-B-NPs remained almost the same. The cell uptake mechanism study demonstrated that the internalization due to the electrostatic interaction would contribute more to the cell uptake when the internalization based on clathrin-mediated endocytosis and other ATP-dependent pathways were blocked. The co-decoration of CS and target ligand is an effective approach for improving the specific cell uptake of NPs.

  20. Alzheimer's therapeutics targeting amyloid beta 1-42 oligomers II: Sigma-2/PGRMC1 receptors mediate Abeta 42 oligomer binding and synaptotoxicity.

    Science.gov (United States)

    Izzo, Nicholas J; Xu, Jinbin; Zeng, Chenbo; Kirk, Molly J; Mozzoni, Kelsie; Silky, Colleen; Rehak, Courtney; Yurko, Raymond; Look, Gary; Rishton, Gilbert; Safferstein, Hank; Cruchaga, Carlos; Goate, Alison; Cahill, Michael A; Arancio, Ottavio; Mach, Robert H; Craven, Rolf; Head, Elizabeth; LeVine, Harry; Spires-Jones, Tara L; Catalano, Susan M

    2014-01-01

    Amyloid beta (Abeta) 1-42 oligomers accumulate in brains of patients with Mild Cognitive Impairment (MCI) and disrupt synaptic plasticity processes that underlie memory formation. Synaptic binding of Abeta oligomers to several putative receptor proteins is reported to inhibit long-term potentiation, affect membrane trafficking and induce reversible spine loss in neurons, leading to impaired cognitive performance and ultimately to anterograde amnesia in the early stages of Alzheimer's disease (AD). We have identified a receptor not previously associated with AD that mediates the binding of Abeta oligomers to neurons, and describe novel therapeutic antagonists of this receptor capable of blocking Abeta toxic effects on synapses in vitro and cognitive deficits in vivo. Knockdown of sigma-2/PGRMC1 (progesterone receptor membrane component 1) protein expression in vitro using siRNA results in a highly correlated reduction in binding of exogenous Abeta oligomers to neurons of more than 90%. Expression of sigma-2/PGRMC1 is upregulated in vitro by treatment with Abeta oligomers, and is dysregulated in Alzheimer's disease patients' brain compared to age-matched, normal individuals. Specific, high affinity small molecule receptor antagonists and antibodies raised against specific regions on this receptor can displace synthetic Abeta oligomer binding to synaptic puncta in vitro and displace endogenous human AD patient oligomers from brain tissue sections in a dose-dependent manner. These receptor antagonists prevent and reverse the effects of Abeta oligomers on membrane trafficking and synapse loss in vitro and cognitive deficits in AD mouse models. These findings suggest sigma-2/PGRMC1 receptors mediate saturable oligomer binding to synaptic puncta on neurons and that brain penetrant, small molecules can displace endogenous and synthetic oligomers and improve cognitive deficits in AD models. We propose that sigma-2/PGRMC1 is a key mediator of the pathological effects of

  1. Alzheimer's therapeutics targeting amyloid beta 1-42 oligomers II: Sigma-2/PGRMC1 receptors mediate Abeta 42 oligomer binding and synaptotoxicity.

    Directory of Open Access Journals (Sweden)

    Nicholas J Izzo

    Full Text Available Amyloid beta (Abeta 1-42 oligomers accumulate in brains of patients with Mild Cognitive Impairment (MCI and disrupt synaptic plasticity processes that underlie memory formation. Synaptic binding of Abeta oligomers to several putative receptor proteins is reported to inhibit long-term potentiation, affect membrane trafficking and induce reversible spine loss in neurons, leading to impaired cognitive performance and ultimately to anterograde amnesia in the early stages of Alzheimer's disease (AD. We have identified a receptor not previously associated with AD that mediates the binding of Abeta oligomers to neurons, and describe novel therapeutic antagonists of this receptor capable of blocking Abeta toxic effects on synapses in vitro and cognitive deficits in vivo. Knockdown of sigma-2/PGRMC1 (progesterone receptor membrane component 1 protein expression in vitro using siRNA results in a highly correlated reduction in binding of exogenous Abeta oligomers to neurons of more than 90%. Expression of sigma-2/PGRMC1 is upregulated in vitro by treatment with Abeta oligomers, and is dysregulated in Alzheimer's disease patients' brain compared to age-matched, normal individuals. Specific, high affinity small molecule receptor antagonists and antibodies raised against specific regions on this receptor can displace synthetic Abeta oligomer binding to synaptic puncta in vitro and displace endogenous human AD patient oligomers from brain tissue sections in a dose-dependent manner. These receptor antagonists prevent and reverse the effects of Abeta oligomers on membrane trafficking and synapse loss in vitro and cognitive deficits in AD mouse models. These findings suggest sigma-2/PGRMC1 receptors mediate saturable oligomer binding to synaptic puncta on neurons and that brain penetrant, small molecules can displace endogenous and synthetic oligomers and improve cognitive deficits in AD models. We propose that sigma-2/PGRMC1 is a key mediator of the pathological

  2. Brain tumor-targeted therapy by systemic delivery of siRNA with Transferrin receptor-mediated core-shell nanoparticles.

    Science.gov (United States)

    Wei, Lin; Guo, Xi-Ying; Yang, Ting; Yu, Min-Zhi; Chen, Da-Wei; Wang, Jian-Cheng

    2016-08-20

    Treatment of brain tumor remains a great challenge worldwide. Development of a stable, safe, and effective siRNA delivery system which is able to cross the impermeable blood-brain barrier (BBB) and target glioma cells is necessary. This study aims to investigate the therapeutic effects of intravenous administration of T7 peptide modified core-shell nanoparticles (named T7-LPC/siRNA NPs) on brain tumors. Layer-by-layer assembling of protamine/chondroitin sulfate/siRNA/cationic liposomes followed by T7 peptide modification has been carried out in order to obtain a targeted siRNA delivery system. In vitro cellular uptake experiments demonstrated a higher intracellular fluorescence intensity of siRNA in brain microvascular endothelial cells (BMVECs) and U87 glioma cells when treated with T7-LPC/siRNA NPs compared with PEG-LPC/siRNA NPs. In the co-culture model of BMVECs and U87 cells, a significant down-regulation of EGFR protein expression occurred in the U87 glioma cells after treatment with the T7-LPC/siEGFR NPs. Moreover, the T7-LPC/siRNA NPs had an advantage in penetrating into a deep region of the tumor spheroid compared with PEG-LPC/siRNA NPs. In vivo imaging revealed that T7-LPC/siRNA NPs accumulated more specifically in brain tumor tissues than the non-targeted NPs. Also, in vivo tumor therapy experiments demonstrated that the longest survival period along with the greatest downregulation of EGFR expression in tumor tissues was observed in mice with an intracranial U87 glioma treated with T7-LPC/siEGFR NPs compared with mice receiving other formulations. Therefore, we believe that these transferrin receptor-mediated core-shell nanoparticles are an important potential siRNA delivery system for brain tumor-targeted therapy.

  3. Enzymatically Modified Low-Density Lipoprotein Promotes Foam Cell Formation in Smooth Muscle Cells via Macropinocytosis and Enhances Receptor-Mediated Uptake of Oxidized Low-Density Lipoprotein.

    Science.gov (United States)

    Chellan, Bijoy; Reardon, Catherine A; Getz, Godfrey S; Hofmann Bowman, Marion A

    2016-06-01

    Enzyme-modified nonoxidized low-density lipoprotein (ELDL) is present in human atherosclerotic lesions. Our objective is to understand the mechanisms of ELDL uptake and its effects on vascular smooth muscle cells (SMC). Transformation of murine aortic SMCs into foam cells in response to ELDL was analyzed. ELDL, but not acetylated or oxidized LDL, was potent in inducing SMC foam cell formation. Inhibitors of macropinocytosis (LY294002, wortmannin, amiloride) attenuated ELDL uptake. In contrast, inhibitors of receptor-mediated endocytosis (dynasore, sucrose) and inhibitor of caveolae-/lipid raft-mediated endocytosis (filipin) had no effect on ELDL uptake in SMC, suggesting that macropinocytosis is the main mechanism of ELDL uptake by SMC. Receptor for advanced glycation end products (RAGE) is not obligatory for ELDL-induced SMC foam cell formation, but primes SMC for the uptake of oxidized LDL in a RAGE-dependent manner. ELDL increased intracellular reactive oxygen species, cytosolic calcium, and expression of lectin-like oxidized LDL receptor-1 in wild-type SMC but not in RAGE(-/-) SMC. The macropinocytotic uptake of ELDL is regulated predominantly by intracellular calcium because ELDL uptake was completely inhibited by pretreatment with the calcium channel inhibitor lacidipine in wild-type and RAGE(-/-) SMC. This is in contrast to pretreatment with PI3 kinase inhibitors which completely prevented ELDL uptake in RAGE(-/-) SMC, but only partially in wild-type SMC. ELDL is highly potent in inducing foam cells in murine SMC. ELDL endocytosis is mediated by calcium-dependent macropinocytosis. Priming SMC with ELDL enhances the uptake of oxidized LDL. © 2016 American Heart Association, Inc.

  4. Pharmacological characterisation of the adenosine receptor mediating increased ion transport in the mouse isolated trachea and the effect of allergen challenge.

    Science.gov (United States)

    Kornerup, Kristin N; Page, Clive P; Moffatt, James D

    2005-04-01

    The effect of adenosine on transepithelial ion transport was investigated in isolated preparations of murine trachea mounted in Ussing chambers. The possible regulation of adenosine receptors in an established model of allergic airway inflammation was also investigated. Mucosally applied adenosine caused increases in short-circuit current (I(SC)) that corresponded to approximately 50% of the response to the most efficacious secretogogue, ATP (delta I(SC) 69.5 +/- 6.7 microA cm2). In contrast, submucosally applied adenosine caused only small (<20%) increases in I(SC), which were not investigated further. The A1-selective (N6-cyclopentyladenosine, CPA, 1 nM-10 microM), A2A-selective (2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxoamido adenosine; CGS 21680; 0.1-100 microM) and A3-selective (1-deoxy-1-[6-[[(3-iodophenyl)-methyl]amino]-9H-purin-9-yl]-N-methyl-beta-D-ribofuranuronamide; IB-MECA; 30 nM-100 microM) adenosine receptor agonists were either equipotent or less potent than adenosine, suggesting that these receptors do not mediate the response to adenosine. The A1 receptor selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 10 nM-1 microM) caused a rightward shift of the adenosine concentration-effect curve only at 1 microM. The mixed A2A/A2B receptor antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) also caused rightward shift of the adenosine concentration-effect curve, again only at micromolar concentrations, suggestive of the involvement of A2B receptors. In preparations from animals sensitised to ovalbumin and challenged over 3 days with aerosol ovalbumin, a decrease in baseline I(SC) was observed and responses to ATP were diminished. Similarly, the amplitude of responses to adenosine were attenuated although there was no change in potency. These results suggest that the A2B receptor mediates the I(SC) response to adenosine in the mouse trachea. This receptor does not appear to be

  5. The differential effects of 5-HT(1A) receptor stimulation on dopamine receptor-mediated abnormal involuntary movements and rotations in the primed hemiparkinsonian rat.

    Science.gov (United States)

    Dupre, Kristin B; Eskow, Karen L; Negron, Giselle; Bishop, Christopher

    2007-07-16

    Serotonin 1A receptor (5-HT(1A)R) agonists have emerged as valuable supplements to l-DOPA therapy, demonstrating that they can decrease side effects and enhance motor function in animal models of Parkinson's disease (PD) and human PD patients. The precise mechanism by which these receptors act remains unknown and there is limited information on how 5-HT(1A)R stimulation impacts striatal dopamine (DA) D1 receptor (D1R) and D2 receptor (D2R) function. The current study examined the effects of 5-HT(1A)R stimulation on DA receptor-mediated behaviors. Male Sprague-Dawley rats were rendered hemiparkinsonian by unilateral 6-OHDA lesions and primed with the D1R agonist SKF81297 (0.8 mg/kg, i.p.) in order to sensitize DA receptors. Using a randomized within subjects design, rats received a first injection of: Vehicle (dH(2)O) or the 5-HT(1A)R agonist +/-8-OH-DPAT (0.1 or 1.0 mg/kg, i.p.), followed by a second injection of: Vehicle (dimethyl sulfoxide), the D1R agonist SKF81297 (0.8 mg/kg, i.p.), the D2R agonist quinpirole (0.2 mg/kg, i.p.), or l-DOPA (12 mg/kg+benserazide, 15 mg/kg, i.p.). On test days, rats were monitored over a 2-h period immediately following the second injection for abnormal involuntary movements (AIMs), analogous to dyskinesia observed in PD patients, and contralateral rotations. The present findings indicate that 5-HT(1A)R stimulation reduces AIMs induced by D1R, D2R and l-DOPA administration while its effects on DA agonist-induced rotations were receptor-dependent, suggesting that direct 5-HT(1A)R and DA receptor interactions may contribute to the unique profile of 5-HT(1A)R agonists for the improvement of PD treatment.

  6. Mode of Action and Human Relevance Analysis for Nuclear Receptor-Mediated Liver Toxicity: A Case Study with Phenobarbital as a Model Constitutive Androstane Receptor (CAR) Activator

    Science.gov (United States)

    The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are key nuclear receptors involved in the regulation of cellular responses. to exposure to many xenobiotics and various physiological processes. Phenobarbital (PB) is a non­ genotoxic i...

  7. Stress-induced release of anterior pituitary hormones: Effect of H3 receptor-mediated inhibition of histaminergic activity or posterior hypothalamic lesion

    DEFF Research Database (Denmark)

    Knigge, U.; Søe-Jensen, P.; Jørgensen, Henrik;

    1999-01-01

    Histamine receptors, corticotropin, *Gb-endorphin, prolactin, adrenal steroids, stress, endotoxin, serotonin......Histamine receptors, corticotropin, *Gb-endorphin, prolactin, adrenal steroids, stress, endotoxin, serotonin...

  8. Genetic engineering, expression, and activity of a chimeric monoclonal antibody-avidin fusion protein for receptor-mediated delivery of biotinylated drugs in humans.

    Science.gov (United States)

    Boado, Ruben J; Zhang, Yufeng; Zhang, Yun; Xia, Chun-fang; Wang, Yuntao; Pardridge, William M

    2008-03-01

    The genetic engineering, expression, and validation of a fusion protein of avidin (AV) and a chimeric monoclonal antibody (mAb) to the human insulin receptor (HIR) is described. The 15 kDa avidin monomer was fused to the carboxyl terminus of the heavy chain of the HIRMAb. The fusion protein heavy chain reacted with antibodies specific for human IgG and avidin, and had the same affinity for binding to the HIR extracellular domain as the original chimeric HIRMAb. The fusion protein qualitatively bound biotinylated ligands, but was secreted fully saturated with biotin by COS cells, owing to the high level of biotin in tissue culture medium. Chinese hamster ovary (CHO) cells were permanently transfected with a tandem vector expressing the fusion protein genes, and high expressing cell lines were isolated by methotrexate amplification and dilutional cloning. The product expressed by CHO cells had high binding to the HIR, and migrated as a homogeneous species in size exclusion HPLC and native polyacrylamide gel electrophoresis. The CHO cells were adapted to a 4 week culture in biotin depleted medium, and the HIRMAb-AV fusion protein expressed under these conditions had 1 unoccupied biotin binding site per molecule, based on a [3H]-biotin ultrafiltration assay. The HIRMAb-AV increased biotin uptake by human cells >15-fold, and mediated the endocytosis of fluorescein-biotin, as demonstrated by confocal microscopy. In summary, the HIRMAb-AV fusion protein is a new drug targeting system for humans that can be adapted to monobiotinylated drugs or nucleic acids.

  9. Regulation of platelet-activating factor (PAF) receptor and PAF receptor-mediated cellular response in Kupffer cells: Effect of vanadate

    Energy Technology Data Exchange (ETDEWEB)

    Chao, W.; Liu, H.; Hanahan, D.J.; Olson, M.S. (Univ. of Texas, San Antonio (United States))

    1991-03-11

    Vanadate is a phosphate analogue which affects phosphate transfer reactions which may be involved in regulatory processes in which tyrosine phosphorylation or dephosphorylation may be an important component. In the present study vanadate decreased the surface expression of PAF receptors and caused tyrosine-phosphorylation in numerous proteins in intact Kupffer cells. The vanadate-induced tyrosine-phosphorylation was inhibited by genistein, a specific tyrosine kinase inhibitor. The EC{sub 50} for the vanadate-initiated decrease in the surface expression of PAF receptors was approximately 0.25 mM, 0.65 mM, and 2 mM, respectively, when the vanadate exposure time was 3 h, 2h, and 1h. As a consequence, PAF-stimulated prostaglandin E{sub 2} (PGE{sub 2}) formation was attenuated in vanadate-treated Kupffer cells. While vanadate itself was found to stimulate PGE{sub 2} production, PAF-stimulated PGE{sub 2}formation was inhibited significantly by genistein. The present study suggests that vanadate stimulated strongly tyrosine-phosphorylation of cellular proteins and decreased the surface expression of PAF receptor in intact Kupffer cells.

  10. Selective 5-HT7 Receptor Activation May Enhance Synaptic Plasticity Through N-methyl-D-aspartate (NMDA) Receptor Activity in the Visual Cortex.

    Science.gov (United States)

    Xiang, Kangjian; Zhao, Xuefei; Li, Youjun; Zheng, Liang; Wang, Jue; Li, Yan-Hai

    2016-01-01

    Serotonin (5-hydroxytryptamine, 5-HT) is an important neurotransmitter that modulates N-methyl-D-aspartate (NMDA) receptor activity by binding to several different 5-HT receptor subtypes. In the present study, we used whole-cell patch-clamp recordings in transverse slice preparations to test the role of 5-HT receptors in modulating the NMDA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) in layer II/III pyramidal neurons of the rat visual cortex. We found that the NMDA receptor-mediated component of mEPSCs could be potentiated by exogenously applied 5-HT. Similar results were obtained by exogenously applied 5-CT or 8-OH-DPAT (the 5-HT1A and 5-HT7 receptor agonist). A specific antagonist for the 5-HT7 receptor, SB-269970, completely blocked the increase in NMDA receptor-mediated component of mEPSCs by 5-CT or 8- OH-DPAT. Moreover, the selective 5-HT1A receptor antagonist, WAY-100135, displayed no influence on the enhancement in NMDA receptor-mediated component of mEPSCs by 5-CT or 8-OHDPAT. These results indicated that the increase in NMDA receptor-mediated component of mEPSCs by 5-HT in layer II/III pyramidal neurons of the young rat visual cortex requires activation of 5-HT7 receptors, but not 5-HT1A receptors. These observations might be clinically relevant to schizophrenia and Alzheimer's disease (AD), where enhancing NMDA receptor-mediated neurotransmission is considered to be a promising strategy for treatment of these diseases.

  11. Molecular pharmacological phenotyping of EBI2. An orphan seven-transmembrane receptor with constitutive activity

    DEFF Research Database (Denmark)

    Rosenkilde, Mette M; Benned-Jensen, Tau; Holst, Peter J

    2006-01-01

    Epstein-Barr virus (EBV)-induced receptor 2 (EBI2) is an orphan seven-transmembrane (7TM) receptor originally identified as the most up-regulated gene (>200-fold) in EBV-infected cells. Here we show that EBI2 signals with constitutive activity through Galpha(i) as determined by a receptor-mediate...

  12. Endocannabinoid CB1 Receptor Mediated Rises in Ca2+ and Depolarization-Induced Suppression of Inhibition within the Laterodorsal Tegmental Nucleus

    DEFF Research Database (Denmark)

    Soni, Neeraj; Kohlmeier, Kristi Anne

    2016-01-01

    Cannabinoid type 1 receptors (CB1Rs) are functionally active within the laterodorsal tegmental nucleus (LDT), which is critically involved in control of rapid eye movement sleep, cortical arousal, and motivated states. To further characterize the cellular consequences of activation of CB1Rs...

  13. Activation of 5-HT1B receptors suppresses low but not high frequency synaptic transmission in the rat subicular cortex in vitro

    NARCIS (Netherlands)

    Boeijinga, PH; Boddeke, HWGM

    1996-01-01

    We have shown previously that activation of 5-HT1B serotonin receptors mediates suppression of the amplitude of evoked potentials in the subiculum [2]. Here we show that after application of 5-HT (10 mu M), excitatory postsynaptic potentials of subicular neurons have reduced amplitudes with no chang

  14. Activation of 5-HT(1B) receptors suppresses low but not high frequency synaptic transmission in the rat subicular cortex in vitro

    NARCIS (Netherlands)

    Boeijinga, P.H.; Boddeke, H.W.G.M.

    1996-01-01

    We have shown previously that activation of 5-HT(1B) serotonin receptors mediates suppression of the amplitude of evoked potentials in the subiculum [2]. Here we show that after application of 5-HT (10 μM), excitatory postsynaptic potentials of subicular neurons have reduced amplitudes with no chang

  15. Spinal 5-HT3 receptors mediate descending facilitation and contribute to behavioral hypersensitivity via a reciprocal neuron-glial signaling cascade

    OpenAIRE

    Guo, Wei; Miyoshi, Kan; Dubner, Ronald; Gu, Ming; Li, Man; Liu, Jian; Yang, Jiale; Zou, Shiping; Ren, Ke; Noguchi, Koichi; Wei, Feng

    2014-01-01

    Background It has been recently recognized that the descending serotonin (5-HT) system from the rostral ventromedial medulla (RVM) in the brainstem and the 5-HT3 receptor subtype in the spinal dorsal horn are involved in enhanced descending pain facilitation after tissue and nerve injury. However, the mechanisms underlying the activation of the 5-HT3 receptor and its contribution to facilitation of pain remain unclear. Results In the present study, activation of spinal 5-HT3 receptors by intr...

  16. A mechanism-based mathematical model of aryl hydrocarbon receptor-mediated CYP1A induction in rats using beta-naphthoflavone as a tool compound.

    Science.gov (United States)

    Chen, Emile P; Chen, Liangfu; Ji, Yan; Tai, Guoying; Wen, Yuan H; Ellens, Harma

    2010-12-01

    β-Naphthoflavone (BNF) is a synthetic flavone that selectively and potently induces CYP1A enzymes via aryl hydrocarbon receptor activation. Mechanism-based mathematical models of CYP1A enzyme induction were developed to predict the time course of enzyme induction and quantitatively evaluate the interrelationship between BNF plasma concentrations, hepatic CYP1A1 and CYP1A2 mRNA levels, and CYP1A enzyme activity in rats in vivo. Male Sprague-Dawley rats received a continuous intravenous infusion of vehicle or 1.5 or 6 mg · kg(-1) · h(-1) BNF for 6 h, with blood and liver sampling. Plasma BNF concentrations were determined by liquid chromatography-tandem mass spectrometry. Hepatic mRNA levels of CYP1A1 and CYP1A2 were determined by TaqMan. Ethoxyresorufin O-deethylation was used to measure the increase in CYP1A enzyme activity as a result of induction. The induction of hepatic CYP1A1/CYP1A2 mRNA and CYP1A activity occurred within 2 h after BNF administration. This caused a rapid increase in metabolic clearance of BNF, resulting in plasma concentrations declining during the infusion. Overall, the enzyme induction models developed in this study adequately captured the time course of BNF pharmacokinetics, CYP1A1/CYP1A2 mRNA levels, and increases in CYP1A enzyme activity data for both dose groups simultaneously. The model-predicted degradation half-life of CYP1A enzyme activity is comparable with previously reported values. The present results also confirm a previous in vitro finding that CYP1A1 is the predominant contributor to CYP1A induction. These physiologically based models provide a basis for predicting drug-induced toxicity in humans from in vitro and preclinical data and can be a valuable tool in drug development.

  17. Corticosterone and dopamine D2/D3 receptors mediate the motivation for voluntary wheel running in C57BL/6J mice.

    Science.gov (United States)

    Ebada, Mohamed Elsaed; Kendall, David A; Pardon, Marie-Christine

    2016-09-15

    Physical exercise can improve cognition but whether this is related to motivation levels is unknown. Voluntary wheel running is a rewarding activity proposed as a model of motivation to exercise. To question the potential effects of exercise motivation on subsequent behaviour, we used a pharmacological approach targeting some reward mechanisms. The stress hormone corticosterone has rewarding effects mediated by activation of low affinity glucocorticoid receptors (GR). To investigate whether corticosterone synthesis motivates exercise via activation of GRs and subsequently, impacts on behaviour, we treated C57BL/6J mice acutely with the inhibitor of corticosterone synthesis metyrapone (35mg/kg) or repeatedly with the GR antagonist mifepristone (30mg/kg) prior to 1-h running wheel sessions. To investigate whether reducing motivation to exercise impacts on behaviour, we antagonised running-induced dopamine D2/D3 receptors activation with sulpiride (25 or 50mg/kg) and assessed locomotor, anxiety-related and memory performance after 20 running sessions over 4 weeks. We found that corticosterone synthesis contributes to running levels, but the maintenance of running behaviour was not mediated by activation of GRs. Intermittent exercise was not associated with changes in behavioural or cognitive performance. The persistent reduction in exercise levels triggered by sulpiride also had limited impact on behavioural performance, although the level of performance for some behaviours was related to the level of exercise. Altogether, these findings indicate that corticosterone and dopamine D2/D3 receptor activation contribute to the motivation for wheel running, but suggest that motivation for exercise is not a sufficient factor to alter behaviour in healthy mice.

  18. Dopamine D1 Receptor-Mediated Transmission Maintains Information Flow Through the Cortico-Striato-Entopeduncular Direct Pathway to Release Movements.

    Science.gov (United States)

    Chiken, Satomi; Sato, Asako; Ohta, Chikara; Kurokawa, Makoto; Arai, Satoshi; Maeshima, Jun; Sunayama-Morita, Tomoko; Sasaoka, Toshikuni; Nambu, Atsushi

    2015-12-01

    In the basal ganglia (BG), dopamine plays a pivotal role in motor control, and dopamine deficiency results in severe motor dysfunctions as seen in Parkinson's disease. According to the well-accepted model of the BG, dopamine activates striatal direct pathway neurons that directly project to the output nuclei of the BG through D1 receptors (D1Rs), whereas dopamine inhibits striatal indirect pathway neurons that project to the external pallidum (GPe) through D2 receptors. To clarify the exact role of dopaminergic transmission via D1Rs in vivo, we developed novel D1R knockdown mice in which D1Rs can be conditionally and reversibly regulated. Suppression of D1R expression by doxycycline treatment decreased spontaneous motor activity and impaired motor ability in the mice. Neuronal activity in the entopeduncular nucleus (EPN), one of the output nuclei of the rodent BG, was recorded in awake conditions to examine the mechanism of motor deficits. Cortically evoked inhibition in the EPN mediated by the cortico-striato-EPN direct pathway was mostly lost during suppression of D1R expression, whereas spontaneous firing rates and patterns remained unchanged. On the other hand, GPe activity changed little. These results suggest that D1R-mediated dopaminergic transmission maintains the information flow through the direct pathway to appropriately release motor actions.

  19. Calmodulin kinase II-dependent transactivation of PDGF receptors mediates astrocytic MMP-9 expression and cell motility induced by lipoteichoic acid

    Directory of Open Access Journals (Sweden)

    Hsieh Hsi-Lung

    2010-11-01

    Full Text Available Abstract Background Lipoteichoic acid (LTA is a component of Gram-positive bacterial cell walls, which has been found to be elevated in cerebrospinal fluid of patients suffering from meningitis. Moreover, matrix metalloproteinases (MMPs, MMP-9 especially, have been observed in patients with brain inflammatory diseases and may contribute to brain disease pathology. However, the molecular mechanisms underlying LTA-induced MMP-9 expression in brain astrocytes remain unclear. Objective The goal of this study was to examine whether LTA-induced cell migration is mediated by calcium/calmodulin (CaM/CaM kinase II (CaMKII-dependent transactivation of the PDGFR pathway in rat brain astrocytes (RBA-1 cells. Methods Expression and activity of MMP-9 induced by LTA was evaluated by zymographic, western blotting, and RT-PCR analyses. MMP-9 regulatory signaling pathways were investigated by treatment with pharmacological inhibitors or using dominant negative mutants or short hairpin RNA (shRNA transfection, and chromatin immunoprecipitation (ChIP-PCR and promoter activity reporter assays. Finally, we determined the cell functional changes by cell migration assay. Results The data show that c-Jun/AP-1 mediates LTA-induced MMP-9 expression in RBA-1 cells. Next, we demonstrated that LTA induces MMP-9 expression via a calcium/CaM/CaMKII-dependent transactivation of PDGFR pathway. Transactivation of PDGFR led to activation of PI3K/Akt and JNK1/2 and then activated c-Jun/AP-1 signaling. Activated-c-Jun bound to the AP-1-binding site of the MMP-9 promoter, and thereby turned on transcription of MMP-9. Eventually, up-regulation of MMP-9 by LTA enhanced cell migration of astrocytes. Conclusions These results demonstrate that in RBA-1 cells, activation of c-Jun/AP-1 by a CaMKII-dependent PI3K/Akt-JNK activation mediated through transactivation of PDGFR is essential for up-regulation of MMP-9 and cell migration induced by LTA. Understanding the regulatory mechanisms

  20. Modulation of EGF receptor-mediated vulva development by the heterotrimeric G-protein Galphaq and excitable cells in C. elegans.

    Science.gov (United States)

    Moghal, Nadeem; Garcia, L Rene; Khan, Liakot A; Iwasaki, Kouichi; Sternberg, Paul W

    2003-10-01

    The extent to which excitable cells and behavior modulate animal development has not been examined in detail. Here, we demonstrate the existence of a novel pathway for promoting vulval fates in C. elegans that involves activation of the heterotrimeric Galphaq protein, EGL-30. EGL-30 acts with muscle-expressed EGL-19 L-type voltage-gated calcium channels to promote vulva development, and acts downstream or parallel to LET-60 (RAS). This pathway is not essential for vulval induction on standard Petri plates, but can be stimulated by expression of activated EGL-30 in neurons, or by an EGL-30-dependent change in behavior that occurs in a liquid environment. Our results indicate that excitable cells and animal behavior can provide modulatory inputs into the effects of growth factor signaling on cell fates, and suggest that communication between these cell populations is important for normal development to occur under certain environmental conditions.

  1. CGRP receptors mediating CGRP-, adrenomedullin- and amylin-induced relaxation in porcine coronary arteries. Characterization with 'Compound 1' (WO98/11128), a non-peptide antagonist

    DEFF Research Database (Denmark)

    Hasbak, P; Sams, A; Schifter, S

    2001-01-01

    1. Calcitonin gene-related peptide (CGRP), amylin and adrenomedullin (AM) belong to the same family of peptides. Accumulating evidence indicate that the calcitonin (CT) receptor, the CT receptor-like receptor (CRLR) and receptor-activity-modifying proteins (RAMPs) form the basis of all the recept......1. Calcitonin gene-related peptide (CGRP), amylin and adrenomedullin (AM) belong to the same family of peptides. Accumulating evidence indicate that the calcitonin (CT) receptor, the CT receptor-like receptor (CRLR) and receptor-activity-modifying proteins (RAMPs) form the basis of all....... The partial porcine mRNA sequences shared 82 - 92% nucleotide identity with human sequences. 3. The human peptides alphaCGRP, betaCGRP, AM and amylin induced relaxation with pEC(50) values of 8.1, 8.1, 6.7 and 6.1 M respectively. 4. The antagonistic properties of a novel non-peptide CGRP antagonist 'Compound...

  2. Escitalopram attenuates β-amyloid-induced tau hyperphosphorylation in primary hippocampal neurons through the 5-HT1A receptor mediated Akt/GSK-3β pathway

    OpenAIRE

    Wang, Yan-Juan; Ren, Qing-Guo; Gong, Wei-Gang; Wu, Di; Tang, Xiang; Li, Xiao-Li; Wu, Fang-Fang; Bai, Feng; Xu, Lin; Zhang, Zhi-Jun

    2016-01-01

    Tau hyperphosphorylation is an important pathological feature of Alzheimer's disease (AD). To investigate whether escitalopram could inhibit amyloid-β (Aβ)-induced tau hyperphosphorylation and the underlying mechanisms, we treated the rat primary hippocampal neurons with Aβ1-42 and examined the effect of escitalopram on tau hyperphosphorylation. Results showed that escitalopram decreased Aβ1–42-induced tau hyperphosphorylation. In addition, escitalopram activated the Akt/GSK-3β pathway, and t...

  3. The long-acting β2 -adrenoceptor agonist, indacaterol, enhances glucocorticoid receptor-mediated transcription in human airway epithelial cells in a gene- and agonist-dependent manner.

    Science.gov (United States)

    Joshi, T; Johnson, M; Newton, R; Giembycz, M A

    2015-05-01

    Inhaled glucocorticoid (ICS)/long-acting β2 -adrenoceptor agonist (LABA) combination therapy is a recommended treatment option for patients with moderate/severe asthma in whom adequate control cannot be achieved by an ICS alone. Previously, we discovered that LABAs can augment dexamethasone-inducible gene expression and proposed that this effect may explain how these two drugs interact to deliver superior clinical benefit. Herein, we extended that observation by analysing, pharmacodynamically, the effect of the LABA, indacaterol, on glucocorticoid receptor (GR)-mediated gene transcription induced by seven ligands with intrinsic activity values that span the spectrum of full agonism to antagonism. BEAS-2B human airway epithelial cells stably transfected with a 2× glucocorticoid response element luciferase reporter were used to model gene transcription together with an analysis of several glucocorticoid-inducible genes. Indacaterol augmented glucocorticoid-induced reporter activation in a manner that was positively related to the intrinsic activity of the GR agonist. This effect was demonstrated by an increase in response maxima without a change in GR agonist affinity or efficacy. Indacaterol also enhanced glucocorticoid-inducible gene expression. However, the magnitude of this effect was dependent on both the GR agonist and the gene of interest. These data suggest that indacaterol activates a molecular rheostat, which increases the transcriptional competency of GR in an agonist- and gene-dependent manner without apparently changing the relationship between fractional GR occupancy and response. These findings provide a platform to rationally design ICS/LABA combination therapy that is based on the generation of agonist-dependent gene expression profiles in target and off-target tissues. © 2015 The British Pharmacological Society.

  4. Adenylyl cyclase type 6 overexpression selectively enhances β-adrenergic and prostacyclin receptor-mediated inhibition of cardiac fibroblast function because of colocalization in lipid rafts

    OpenAIRE

    Liu, Xiaoqiu; Thangavel, Muthusamy; Sun, Shu Qiang; Kaminsky, Joseph; Mahautmr, Penden; Stitham, Jeremiah; Hwa, John; Ostrom, Rennolds S.

    2007-01-01

    Cardiac fibroblasts produce and degrade extracellular matrix and are critical in regulating cardiac remodeling and hypertrophy. Fibroblasts are activated by factors such as transforming growth factor β and inhibited by agents that elevate 3′,5′-cyclic adenosine monophosphate (cAMP) levels. cAMP signal generation and response is known to be compartmentalized in many cell types in part through the colocalization of receptors and specific adenylyl cyclase isoforms in lipid rafts and caveolae. Th...

  5. Ghrelin receptors mediate ghrelin-induced excitation of agouti-related protein/neuropeptide Y but not pro-opiomelanocortin neurons.

    Science.gov (United States)

    Chen, Shao-Rui; Chen, Hong; Zhou, Jing-Jing; Pradhan, Geetali; Sun, Yuxiang; Pan, Hui-Lin; Li, De-Pei

    2017-08-01

    Ghrelin increases food intake and body weight by stimulating orexigenic agouti-related protein (AgRP)/neuropeptide Y (NPY) neurons and inhibiting anorexic pro-opiomelanocortin (POMC) neurons in the hypothalamus. Growth hormone secretagogue receptor (Ghsr) mediates the effect of ghrelin on feeding behavior and energy homeostasis. However, the role of Ghsr in the ghrelin effect on these two populations of neurons is unclear. We hypothesized that Ghsr mediates the effect of ghrelin on AgRP and POMC neurons. In this study, we determined whether Ghsr similarly mediates the effects of ghrelin on AgRP/NPY and POMC neurons using cell type-specific Ghsr-knockout mice. Perforated whole-cell recordings were performed on green fluorescent protein-tagged AgRP/NPY and POMC neurons in the arcuate nucleus in hypothalamic slices. In Ghsr(+/+) mice, ghrelin (100 nM) significantly increased the firing activity of AgRP/NPY neurons but inhibited the firing activity of POMC neurons. In Ghsr(-/-) mice, the excitatory effect of ghrelin on AgRP/NPY neurons was abolished. Ablation of Ghsr also eliminated ghrelin-induced increases in the frequency of GABAergic inhibitory postsynaptic currents of POMC neurons. Strikingly, ablation of Ghsr converted the ghrelin effect on POMC neurons from inhibition to excitation. Des-acylated ghrelin had no such effect on POMC neurons in Ghsr(-/-) mice. In both Ghsr(+/+) and Ghsr(-/-) mice, blocking GABAA receptors with gabazine increased the basal firing activity of POMC neurons, and ghrelin further increased the firing activity of POMC neurons in the presence of gabazine. Our findings provide unequivocal evidence that Ghsr is essential for ghrelin-induced excitation of AgRP/NPY neurons. However, ghrelin excites POMC neurons through an unidentified mechanism that is distinct from conventional Ghsr. © 2017 International Society for Neurochemistry.

  6. Control of Toll-like receptor-mediated T cell-independent type 1 antibody responses by the inducible nuclear protein IκB-ζ.

    Science.gov (United States)

    Hanihara-Tatsuzawa, Fumito; Miura, Hanae; Kobayashi, Shuhei; Isagawa, Takayuki; Okuma, Atsushi; Manabe, Ichiro; MaruYama, Takashi

    2014-11-07

    Antibody responses have been classified as being either T cell-dependent or T cell-independent (TI). TI antibody responses are further classified as being either type 1 (TI-1) or type 2 (TI-2), depending on their requirement for B cell-mediated antigen receptor signaling. Although the mechanistic basis of antibody responses has been studied extensively, it remains unclear whether different antibody responses share similarities in their transcriptional regulation. Here, we show that mice deficient in IκB-ζ, specifically in their B cells, have impaired TI-1 antibody responses but normal T cell-dependent and TI-2 antibody responses. The absence of IκB-ζ in B cells also impaired proliferation triggered by Toll-like receptor (TLR) activation, plasma cell differentiation, and class switch recombination (CSR). Mechanistically, IκB-ζ-deficient B cells could not induce TLR-mediated induction of activation-induced cytidine deaminase (AID), a class-switch DNA recombinase. Retroviral transduction of AID in IκB-ζ-deficient B cells restored CSR activity. Furthermore, acetylation of histone H3 in the vicinity of the transcription start site of the gene that encodes AID was reduced in IκB-ζ-deficient B cells relative to IκB-ζ-expressing B cells. These results indicate that IκB-ζ regulates TLR-mediated CSR by inducing AID. Moreover, IκB-ζ defines differences in the transcriptional regulation of different antibody responses.

  7. Design of cholesterol arabinogalactan anchored liposomes for asialoglycoprotein receptor mediated targeting to hepatocellular carcinoma: In silico modeling, in vitro and in vivo evaluation.

    Science.gov (United States)

    Pathak, Pankaj; Dhawan, Vivek; Magarkar, Aniket; Danne, Reinis; Govindarajan, Srinath; Ghosh, Sandipto; Steiniger, Frank; Chaudhari, Pradip; Gopal, Vijaya; Bunker, Alex; Róg, Tomasz; Fahr, Alfred; Nagarsenker, Mangal

    2016-07-25

    We have developed active targeting liposomes to deliver anticancer agents to ASGPR which will contribute to effective treatment of hepatocellular carcinoma. Active targeting is achieved through polymeric ligands on the liposome surface. The liposomes were prepared using reverse phase evaporation method and doxorubicin hydrocholoride, a model drug, was loaded using the ammonium sulphate gradient method. Liposomes loaded with DOX were found to have a particle size of 200nm with more than 90% entrapment efficiency. Systems were observed to release the drug in a sustained manner in acidic pH in vitro. Liposomes containing targeting ligands possessed greater and selective toxicity to ASGPR positive HepG2 cell lines due to specific ligand receptor interaction. Bio-distribution studies revealed that liposomes were concentrated in the liver even after 3h of administration, thus providing conclusive evidence of targeting potential for formulated nanosystems. Tumor regression studies indicated greater tumor suppression with targeted liposomes thereby establishing superiority of the liposomal system. In this work, we used a novel methodology to guide the determination of the optimal composition of the targeting liposomes: molecular dynamics (MD) simulation that aided our understanding of the behaviour of the ligand within the bilayer. This can be seen as a demonstration of the utility of this methodology as a rational design tool for active targeting liposome formulation.

  8. Aryl hydrocarbon receptor-mediated up-regulation of ATP-driven xenobiotic efflux transporters at the blood-brain barrier.

    Science.gov (United States)

    Wang, Xueqian; Hawkins, Brian T; Miller, David S

    2011-02-01

    Many widespread and persistent organic pollutants, e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), activate the aryl hydrocarbon receptor (AhR), causing it to translocate to the cell nucleus, where it transactivates target genes. AhR's ability to target the blood-brain barrier is essentially unexplored. We show here that exposing isolated rat brain capillaries to 0.05-0.5 nM TCDD roughly doubled transport activity and protein expression of P-glycoprotein, an ATP-driven drug efflux pump and a critical determinant of drug entry into the CNS. These effects were abolished by actinomycin D or cycloheximide or by the AhR antagonists resveratrol and α-naphthoflavone. Brain capillaries from TCDD-dosed rats (1-5 μg/kg, i.p.) exhibited increased transport activity and protein expression of 3 xenobiotic efflux pumps, P-glycoprotein, multidrug resistance-associated protein 2, and breast cancer resistance polypeptide, as well as expression of Cyp1a1 and Cyp1b1, both AhR target genes. Consistent with increased P-glycoprotein expression in capillaries from TCDD-dosed rats, in situ brain perfusion indicated significantly reduced brain accumulation of verapamil, a P-glycoprotein substrate. These findings suggest a new paradigm for the field of environmental toxicology: toxicants acting through AhR to target xenobiotic efflux transporters at the blood-brain barrier and thus reduce brain accumulation of CNS-acting therapeutic drugs.

  9. The GLP-1 Receptor Agonist Exendin-4 and Diazepam Differentially Regulate GABAA Receptor-Mediated Tonic Currents in Rat Hippocampal CA3 Pyramidal Neurons.

    Directory of Open Access Journals (Sweden)

    Sergiy V Korol

    Full Text Available Glucagon-like peptide-1 (GLP-1 is a metabolic hormone that is secreted in a glucose-dependent manner and enhances insulin secretion. GLP-1 receptors are also found in the brain where their signalling affects neuronal activity. We have previously shown that the GLP-1 receptor agonists, GLP-1 and exendin-4 enhanced GABA-activated synaptic and tonic currents in rat hippocampal CA3 pyramidal neurons. The hippocampus is the centre for memory and learning and is important for cognition. Here we examined if exendin-4 similarly enhanced the GABA-activated currents in the presence of the benzodiazepine diazepam. In whole-cell recordings in rat brain slices, diazepam (1 μM, an allosteric positive modulator of GABAA receptors, alone enhanced the spontaneous inhibitory postsynaptic current (sIPSC amplitude and frequency by a factor of 1.3 and 1.6, respectively, and doubled the tonic GABAA current normally recorded in the CA3 pyramidal cells. Importantly, in the presence of exendin-4 (10 nM plus diazepam (1 μM, only the tonic but not the sIPSC currents transiently increased as compared to currents recorded in the presence of diazepam alone. The results suggest that exendin-4 potentiates a subpopulation of extrasynaptic GABAA receptors in the CA3 pyramidal neurons.

  10. Novel positive regulatory role for the SPL6 transcription factor in the N TIR-NB-LRR receptor-mediated plant innate immunity.

    Directory of Open Access Journals (Sweden)

    Meenu S Padmanabhan

    2013-03-01

    Full Text Available Following the recognition of pathogen-encoded effectors, plant TIR-NB-LRR immune receptors induce defense signaling by a largely unknown mechanism. We identify a novel and conserved role for the SQUAMOSA PROMOTER BINDING PROTEIN (SBP-domain transcription factor SPL6 in enabling the activation of the defense transcriptome following its association with a nuclear-localized immune receptor. During an active immune response, the Nicotiana TIR-NB-LRR N immune receptor associates with NbSPL6 within distinct nuclear compartments. NbSPL6 is essential for the N-mediated resistance to Tobacco mosaic virus. Similarly, the presumed Arabidopsis ortholog AtSPL6 is required for the resistance mediated by the TIR-NB-LRR RPS4 against Pseudomonas syringae carrying the avrRps4 effector. Transcriptome analysis indicates that AtSPL6 positively regulates a subset of defense genes. A pathogen-activated nuclear-localized TIR-NB-LRR like N can therefore regulate defense genes through SPL6 in a mechanism analogous to the induction of MHC genes by mammalian immune receptors like CIITA and NLRC5.

  11. Capsular Polysaccharide is a Main Component of Mycoplasma ovipneumoniae in the Pathogen-Induced Toll-Like Receptor-Mediated Inflammatory Responses in Sheep Airway Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Zhongjia Jiang

    2017-01-01

    Full Text Available Mycoplasma ovipneumoniae (M. ovipneumoniae is characterized as an etiological agent of primary atypical pneumonia that specifically infects sheep and goat. In an attempt to better understand the pathogen-host interaction between the invading M. ovipneumoniae and airway epithelial cells, we investigated the host inflammatory responses against capsular polysaccharide (designated as CPS of M. ovipneumoniae using sheep bronchial epithelial cells cultured in an air-liquid interface (ALI model. Results showed that CPS derived from M. ovipneumoniae could activate toll-like receptor- (TLR- mediated inflammatory responses, along with an elevated expression of nuclear factor kappa B (NF-κB, activator protein-1 (AP-1, and interferon regulatory factor 3 (IRF3 as well as various inflammatory-associated mediators, representatively including proinflammatory cytokines, such as IL1β, TNFα, and IL8, and anti-inflammatory cytokines such as IL10 and TGFβ of TLR signaling cascade. Mechanistically, the CPS-induced inflammation was TLR initiated and was mediated by activations of both MyD88-dependent and MyD88-independent signaling pathways. Of importance, a blockage of CPS with specific antibody led a significant reduction of M. ovipneumoniae-induced inflammatory responses in sheep bronchial epithelial cells. These results suggested that CPS is a key virulent component of M. ovipneumoniae, which may play a crucial role in the inflammatory response induced by M. ovipneumoniae infections.

  12. The P2Y2 receptor mediates uptake of matrix-retained and aggregated low density lipoprotein in primary vascular smooth muscle cells

    Science.gov (United States)

    Dissmore, Tixieanna; Seye, Cheikh I.; Medeiros, Denis M.; Weisman, Gary A.; Bardford, Barry; Mamedova, Laman

    2016-01-01

    Background and aims The internalization of aggregated low-density lipoproteins (agLDL) mediated by low-density lipoprotein receptor related protein (LRP1) may involve the actin cytoskeleton in ways that differ from the endocytosis of soluble LDL by the LDL receptor (LDLR). This study aims to define novel mechanisms of agLDL uptake through modulation of the actin cytoskeleton, to identify molecular targets involved in foam cell formation in vascular smooth muscle cells (VSMCs). The critical observation that formed the basis for these studies is that under pathophysiological conditions, nucleotide release from blood-derived and vascular cells activates SMC P2Y2 receptors (P2Y2Rs) leading to rearrangement of the actin cytoskeleton and cell motility. Therefore, we tested the hypothesis that P2Y2R activation mediates agLDL uptake by VSMCs. Methods Primary VSMCs were isolated from aortas of wild type (WT) C57BL/6 and.P2Y2R−/− mice to investigate whether P2Y2R activation modulates LRP1 expression. Cells were transiently transfected with cDNA encoding a hemagglutinin-tagged (HA-tagged) WT P2Y2R, or a mutant P2Y2R that unlike the WT P2Y2R does not bind the cytoskeletal actin-binding protein filamin-A (FLN-A). Results P2Y2R activation significantly increased agLDL uptake, and LRP1 mRNA expression decreased in P2Y2R−/− VSMCs versus WT. SMCs, expressing P2Y2R defective in FLN-A binding, exhibit 3-fold lower LDLR expression levels than SMCs expressing WT P2Y2R, while cells transfected with WT P2Y2R show greater agLDL uptake in both WT and P2Y2R−/− VSMCs versus cells transfected with the mutant P2Y2R. Conclusions Together, these results show that both LRP1 and LDLR expression and agLDL uptake are regulated by P2Y2R in VSMCs, and that agLDL uptake due to P2Y2R activation is dependent upon cytoskeletal reorganization mediated by P2Y2R binding to FLN-A. PMID:27522265

  13. QSAR modeling of mono- and bis-quaternary ammonium salts that act as antagonists at neuronal nicotinic acetylcholine receptors mediating dopamine release.

    Science.gov (United States)

    Zheng, Fang; Bayram, Ersin; Sumithran, Sangeetha P; Ayers, Joshua T; Zhan, Chang-Guo; Schmitt, Jeffrey D; Dwoskin, Linda P; Crooks, Peter A

    2006-05-01

    Back-propagation artificial neural networks (ANNs) were trained on a dataset of 42 molecules with quantitative IC50 values to model structure-activity relationships of mono- and bis-quaternary ammonium salts as antagonists at neuronal nicotinic acetylcholine receptors (nAChR) mediating nicotine-evoked dopamine release. The ANN QSAR models produced a reasonable level of correlation between experimental and calculated log(1/IC50) (r2=0.76, r(cv)2=0.64). An external test for the models was performed on a dataset of 18 molecules with IC50 values >1 microM. Fourteen of these were correctly classified. Classification ability of various models, including self-organizing maps (SOM), for all 60 molecules was also evaluated. A detailed analysis of the modeling results revealed the following relative contributions of the used descriptors to the trained ANN QSAR model: approximately 44.0% from the length of the N-alkyl chain attached to the quaternary ammonium head group, approximately 20.0% from Moriguchi octanol-water partition coefficient of the molecule, approximately 13.0% from molecular surface area, approximately 12.6% from the first component shape directional WHIM index/unweighted, approximately 7.8% from Ghose-Crippen molar refractivity, and 2.6% from the lowest unoccupied molecular orbital energy. The ANN QSAR models were also evaluated using a set of 13 newly synthesized compounds (11 biologically active antagonists and two biologically inactive compounds) whose structures had not been previously utilized in the training set. Twelve among 13 compounds were predicted to be active which further supports the robustness of the trained models. Other insights from modeling include a structural modification in the bis-quinolinium series that involved replacing the 5 and/or 8 as well as the 5' and/or 8' carbon atoms with nitrogen atoms, predicting inactive compounds. Such data can be effectively used to reduce synthetic and in vitro screening activities by eliminating

  14. Phosphorylation substrates for protein kinase C in intact pituitary cells: characterization of a receptor-mediated event using novel gonadotropin-releasing hormone analogues

    Energy Technology Data Exchange (ETDEWEB)

    Strulovici, B.; Tahilramani, R.; Nestor, J.J. Jr.

    1987-09-22

    The involvement of protein kinase C in the signal transduction of gonadotropin-releasing hormone (GnRH) action was investigated with a GnRH superagonist, partial agonists, and antagonists in intact rat pituitary cells. Exposure of /sup 32/P-labeled cells to GnRH or to the superagonist (D-Nal(2)/sup 6/)GnRH induced the enhanced phosphorylation of 42-, 34-, 11-, and 10-kDa proteins and the dephosphorylation of a 15-kDa protein as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/autoradiography. This effect was blocked in a dose-dependent manner by potent GnRG antagonists. Downregulation of protein kinase C by prolonged incubation of the pituitary cells with high concentrations of active phorbol esters abolished protein kinase C activity and also prevented the phosphorylation induced by GnRN, or (D-Nal(2)/sup 6/)GnRH. The same effect was obtained by preincubating the cells with the protein kinase C inhibitor H-7. In this study the authors identify for the first time physiological substrates for protein kinase C in intact pituitary cells. They demonstrate a close quantitative correlation between the extent of translocation of protein kinase C, levels of phosphorylation of specific substrates in the intact cells, and the biological activity of the GnRH analogues with varying affinity for the GnRH receptor. These data strengthen the contention that the physiological effects of GnRH are primarily mediated via the phosphatidylinositol/Ca/sup 2 +/ signal transfer system and represent a first step toward defining the physiological substrates of protein kinase C and their role in the cascade of events that starts upon binding of GnRH to its receptor.

  15. Anandamide Suppresses Proinflammatory T Cell Responses In Vitro through Type-1 Cannabinoid Receptor-Mediated mTOR Inhibition in Human Keratinocytes.

    Science.gov (United States)

    Chiurchiù, Valerio; Rapino, Cinzia; Talamonti, Emanuela; Leuti, Alessandro; Lanuti, Mirko; Gueniche, Audrey; Jourdain, Roland; Breton, Lionel; Maccarrone, Mauro

    2016-11-01

    The endocannabinoid system comprises cannabinoid receptors 1 and 2 (CB1 and CB2), their endogenous ligands, anandamide (AEA) and 2-arachidonoylglycerol, and metabolic enzymes of these ligands. The endocannabinoid system has recently been implicated in the regulation of various pathophysiological processes of the skin that include immune competence and/or tolerance of keratinocytes, the disruption of which might promote the development of skin diseases. Recent evidence showed that CB1 in keratinocytes limits the secretion of proinflammatory chemokines, suggesting that this receptor might also regulate T cell dependent inflammatory diseases of the skin. In this article, we sought to investigate the cytokine profile of IFN-γ-activated keratinocytes, and found that CB1 activation by AEA suppressed production and release of signature TH1- and TH17-polarizing cytokines, IL-12 and IL-23, respectively. We also set up cocultures between a conditioned medium of treated keratinocytes and naive T cells to disclose the molecular details that regulate the activation of highly proinflammatory TH1 and TH17 cells. AEA-treated keratinocytes showed reduced an induction of IFN-γ-producing TH1 and IL-17-producing TH17 cells, and these effects were reverted by pharmacological inhibition of CB1 Further analyses identified mammalian target of rapamycin as a proinflammatory signaling pathway regulated by CB1, able to promote either IL-12 and IL-23 release from keratinocytes or TH1 and TH17 polarization. Taken together, these findings demonstrate that AEA suppresses highly pathogenic T cell subsets through CB1-mediated mammalian target of rapamycin inhibition in human keratinocytes. Thus, it can be speculated that the latter pathway might be beneficial to the physiological function of the skin, and can be targeted toward inflammation-related skin diseases. Copyright © 2016 by The American Association of Immunologists, Inc.

  16. Pre-contraction with the thromboxane-mimetic U46619 enhances P2X receptor-mediated contractions in isolated porcine splenic artery.

    Science.gov (United States)

    Roberts, R E

    2012-06-01

    We have previously demonstrated that the thromboxane-mimetic U46619 enhances α(2)-adrenoceptor-mediated contractions through increased activation of extracellular signal-regulated kinase (ERK). In this study, we determined whether U46619 also enhances P2X-mediated contractions through the same pathway. Segments of porcine splenic artery were mounted in isolated tissue baths. Tissues were pre-contracted with U46619 to 10-20% of the response to 60 mM KCl prior to addition of α,β-methylene ATP (P2X receptor agonist). The effect of inhibition of ERK activation with the mitogen-activated protein (MAP)/ERK kinase inhibitor PD98059 (50 μM), Rho kinase inhibition with Y27632 (10 μM), p38 MAP kinase with SB203580 (10 μM) or L-type calcium channels with nifedipine (1 μM) on both the direct and enhanced contractions was then determined. U46619 enhanced the contractions to α,β-methylene ATP. Although PD98059 inhibited the direct contractions to α,β-methylene ATP, it had no effect on the U46619-enhanced contractions. Similarly, Y27632 and SB203580 inhibited the direct contractions to α,β-methylene ATP, but had no effect on the enhanced contractions. Nifedipine inhibited the responses to α,β-methylene ATP in the absence and presence of U46619. This study demonstrates that pre-contraction with U46619 enhances P2X-mediated contractions in the porcine splenic artery through a mechanism independent of ERK, Rho kinase and p38 MAP kinase. Further studies are required to determine the exact mechanism involved.

  17. Adenylyl cyclase type 6 overexpression selectively enhances beta-adrenergic and prostacyclin receptor-mediated inhibition of cardiac fibroblast function because of colocalization in lipid rafts.

    Science.gov (United States)

    Liu, Xiaoqiu; Thangavel, Muthusamy; Sun, Shu Qiang; Kaminsky, Joseph; Mahautmr, Penden; Stitham, Jeremiah; Hwa, John; Ostrom, Rennolds S

    2008-06-01

    Cardiac fibroblasts produce and degrade extracellular matrix and are critical in regulating cardiac remodeling and hypertrophy. Fibroblasts are activated by factors such as transforming growth factor beta and inhibited by agents that elevate 3',5'-cyclic adenosine monophosphate (cAMP) levels. cAMP signal generation and response is known to be compartmentalized in many cell types in part through the colocalization of receptors and specific adenylyl cyclase isoforms in lipid rafts and caveolae. The present study sought to define the localization of key G protein-coupled receptors with adenylyl cyclase type 6 (AC6) in lipid rafts of rat cardiac fibroblasts and to determine if this colocalization was functionally relevant. We found that cardiac fibroblasts produce cAMP in response to agonists for beta-adrenergic (isoproterenol), prostaglandin EP2 (butaprost), adenosine (adenosine-5'-N-ethylcarboxamide, NECA), and prostacyclin (beraprost) receptors. Overexpression of AC6 increased cAMP production stimulated by isoproterenol and beraprost but not by butaprost or NECA. A key function of fibroblasts is the production of collagen. Isoproterenol- and beraprostmediated inhibition of collagen synthesis was also enhanced by AC6 overexpression, while inhibition by butaprost and NECA were unaltered. Lipid raft fractions from cardiac fibroblasts contain the preponderance of beta-adrenergic receptors and AC6 but exclude EP2 receptors. While we could not determine the localization of native prostacyclin receptors, we were able to determine that epitope-tagged prostanoid IP receptors (IPR) expressed in COS7 cells did localize, in part, in lipid raft fractions. These findings indicate that IP receptors are expressed in lipid rafts and can activate raft-localized AC isoforms. AC6 is completely compartmentized in lipid raft domains where it is activated solely by coresident G protein-coupled receptors to regulate cardiac fibroblast function.

  18. Folate Receptor-Mediated Enhanced and Specific Delivery of Far-Red Light-Activatable Prodrugs of Combretastatin A-4 to FR-Positive Tumor

    OpenAIRE

    Nkepang, Gregory; Bio, Moses; Rajaputra, Pallavi; Awuah, Samuel G.; You, Youngjae

    2014-01-01

    We examined the concept of a novel prodrug strategy in which anticancer drug can be locally released by visible/near IR light, taking advantage of the photodynamic process and photo-unclick chemistry. Our most recently formulated prodrug of combretastatin A-4, Pc-(L-CA4)2, showed multifunctionality for fluorescence imaging, light-activated drug release, and the combined effects of PDT and local chemotherapy. In this formulation, L is a singlet oxygen cleavable linker. Here, we advanced this m...

  19. Ethanol exposure during the third trimester equivalent does not affect GABAA or AMPA receptor-mediated spontaneous synaptic transmission in rat CA3 pyramidal neurons

    OpenAIRE

    Baculis, Brian Charles; Valenzuela, Carlos Fernando

    2015-01-01

    Background Ethanol exposure during the rodent equivalent to the 3rd trimester of human pregnancy (i.e., first 1–2 weeks of neonatal life) has been shown to produce structural and functional alterations in the CA3 hippocampal sub-region, which is involved in associative memory. Synaptic plasticity mechanisms dependent on retrograde release of brain-derived neurotrophic factor (BDNF) driven by activation of L-type voltage-gated Ca2+ channels (L-VGCCs) are thought to play a role in stabilization...

  20. cAMP-mediated beta-adrenergic signaling negatively regulates Gq-coupled receptor-mediated fetal gene response in cardiomyocytes.

    Science.gov (United States)

    Patrizio, Mario; Vago, Valerio; Musumeci, Marco; Fecchi, Katia; Sposi, Nadia Maria; Mattei, Elisabetta; Catalano, Liviana; Stati, Tonino; Marano, Giuseppe

    2008-12-01

    The treatment with beta-blockers causes an enhancement of the norepinephrine-induced fetal gene response in cultured cardiomyocytes. Here, we tested whether the activation of cAMP-mediated beta-adrenergic signaling antagonizes alpha(1)-adrenergic receptor (AR)-mediated fetal gene response. To address this question, the fetal gene program, of which atrial natriuretic peptide (ANP) and the beta-isoform of myosin heavy chain are classical members, was induced by phenylephrine (PE), an alpha(1)-AR agonist. In cultured neonatal rat cardiomyocytes, we found that stimulation of beta-ARs with isoproterenol, a beta-AR agonist, inhibited the fetal gene expression induced by PE. Similar results were also observed when cardiomyocytes were treated with forskolin (FSK), a direct activator of adenylyl cyclase, or 8-CPT-6-Phe-cAMP, a selective activator of protein kinase A (PKA). Conversely, the PE-induced fetal gene expression was further upregulated by H89, a selective PKA inhibitor. To evaluate whether these results could be generalized to Gq-mediated signaling and not specifically to alpha(1)-ARs, cardiomyocytes were treated with prostaglandin F(2)alpha, another Gq-coupled receptor agonist, which is able to promote fetal gene expression. This treatment caused an increase of both ANP mRNA and protein levels, which was almost completely abolished by FSK treatment. The capability of beta-adrenergic signaling to regulate the fetal gene expression was also evaluated in vivo conditions by using beta1- and beta2-AR double knockout mice, in which the predominant cardiac beta-AR subtypes are lacking, or by administering isoproterenol (ISO), a beta-AR agonist, at a subpressor dose. A significant increase of the fetal gene expression was found in beta(1)- and beta(2)-AR gene deficient mice. Conversely, we found that ANP, beta-MHC and skACT mRNA levels were significantly decreased in ISO-treated hearts. Collectively, these data indicate that cAMP-mediated beta-adrenergic signaling

  1. Role of G3BP1 in glucocorticoid receptor-mediated microRNA-15b and microRNA-23a biogenesis in endothelial cells

    KAUST Repository

    Kwok, Hoi-Hin

    2017-05-18

    MicroRNAs (miRNAs) are a family of non-coding RNAs that play crucial roles in regulating various normal cellular responses. Recent studies revealed that the canonical miRNA biogenesis pathway is subject to sophisticated regulation. Hormonal control of miRNA biogenesis by androgen and estrogen has been demonstrated, but the direct effects of the glucocorticoid receptor (GR) on miRNA biogenesis are unknown. This study revealed the role of GR in miRNA maturation. We showed that two GR agonists, dexamethasone and ginsenoside-Rg1 rapidly suppressed the expression of mature miR-15b, miR-23a, and miR-214 in human endothelial cells. RNA pulldown coupled with proteomic analysis identified GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1) as one of the RNA-binding proteins mediating GR-regulated miRNA maturation. Activated GR induced phosphorylation of v-AKT Murine Thymoma Viral Oncogene Homologue (AKT) kinase, which in turn phosphorylated and promoted nuclear translocation of G3BP1. The nuclear G3BP1 bound to the G3BP1 consensus sequence located on primary miR-15b~16-2 and miR-23a~27a~24-2 to inhibit their maturation. The findings from this study have advanced our understanding of the non-genomic effects of GR in the vascular system.

  2. Aryl hydrocarbon receptor-mediated toxic potency of dissolved lipophilic organic contaminants collected from Lincoln Creek, Milwaukee, Wisconsin, USA, to PLHC-1 (Poeciliopsis lucida) fish hepatoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Villeneuve, D.L.; Crunkilton, R.L.; DeVita, W.M. [Univ. of Wisconsin, Stevens Point, WI (United States)

    1997-05-01

    Lincoln Creek is a severely degraded urban stream located in Milwaukee County, Wisconsin, USA. As part of a comprehensive study on effects of urban storm water runoff on the stream biota, an in vitro bioassay with PLHC-1 (Poeciliopsis lucida) fish hepatoma cells was used to assess potential toxic potency of aryl hydrocarbon receptor (AhR)-active compounds, collected by semipermeable membrane devices (SPMDs) exposed to Lincoln Creek water. Dialysates from SPMDs exposed to Lincoln Creek water caused marked cytochrome P4501A induction in PLHC-1. Toxic potency of dialysates, expressed as bioassay-derived 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TCDD-EQ) ranged from 1,300 to 6,600 pg TCDD-EQ/g SPMD for 14-d exposures. Dialysates from SPMDs exposed to stream water at base flow had potencies consistently lower than those exposed to storm-flow (high-flow) events that occurred during the same 14-d period. Polychlorinated biphenyls were not detectable in the dialysates. Gas chromatography-mass spectrometry analysis identified polycyclic aromatic hydrocarbons (PAHs) as major contaminants in the dialysates. A log-log correlation of total PAHs and TCDD-EQ yielded an r{sup 2} of 0.802. Empirical evidence suggests that AhR-active PAHs can account for about 20 to 50% of the potency observed.

  3. Red blood cells of sickle cell disease patients exhibit abnormally high abundance of N-methyl D-aspartate receptors mediating excessive calcium uptake.

    Science.gov (United States)

    Hänggi, Pascal; Makhro, Asya; Gassmann, Max; Schmugge, Markus; Goede, Jeroen S; Speer, Oliver; Bogdanova, Anna

    2014-10-01

    Recently we showed that N-methyl D-aspartate receptors (NMDARs) are expressed in erythroid precursors (EPCs) and present in the circulating red blood cells (RBCs) of healthy humans, regulating intracellular Ca(2+) in these cells. This study focuses on investigating the possible role of NMDARs in abnormally high Ca(2+) permeability in the RBCs of patients with sickle cell disease (SCD). Protein levels of the NMDAR subunits in the EPCs of SCD patients did not differ from those in EPCs of healthy humans. However, the number and activity of the NMDARs in circulating SCD-RBCs was substantially up-regulated, being particularly high during haemolytic crises. The number of active NMDARs correlated negatively with haematocrit and haemoglobin levels in the blood of SCD patients. Calcium uptake via these non-selective cation channels was induced by RBC treatment with glycine, glutamate and homocysteine and was facilitated by de-oxygenation of SCD-RBCs. Oxidative stress and RBC dehydration followed receptor stimulation and Ca(2+) uptake. Inhibition of the NMDARs with an antagonist memantine caused re-hydration and largely prevented hypoxia-induced sickling. The EPCs of SCD patients showed higher tolerance to memantine than those of healthy subjects. Consequently, NMDARs in the RBCs of SCD patients appear to be an attractive target for pharmacological intervention.

  4. Pancreatic adenocarcinoma upregulated factor (PAUF) confers resistance to pancreatic cancer cells against oncolytic parvovirus H-1 infection through IFNA receptor-mediated signaling

    Energy Technology Data Exchange (ETDEWEB)

    Kaowinn, Sirichat; Cho, Il-Rae; Moon, Jeong; Jun, Seung Won; Kim, Chang Seok [BK21+, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-736 (Korea, Republic of); Kang, Ho Young [Department of Microbiology, Pusan National University, Busan 609-736 (Korea, Republic of); Kim, Manbok [Department of Medical Science, Dankook University College of Medicine, Cheonan 330-714 (Korea, Republic of); Koh, Sang Seok [Department of Biological Sciences, Dong-A University, Busan 604-714 (Korea, Republic of); Chung, Young-Hwa, E-mail: younghc@pusan.ac.kr [BK21+, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-736 (Korea, Republic of)

    2015-04-03

    Pancreatic adenocarcinoma upregulated factor (PAUF), a novel oncogene, plays a crucial role in the development of pancreatic cancer, including its metastasis and proliferation. Therefore, PAUF-expressing pancreatic cancer cells could be important targets for oncolytic virus-mediated treatment. Panc-1 cells expressing PAUF (Panc-PAUF) showed relative resistance to parvovirus H-1 infection compared with Panc-1 cells expressing an empty vector (Panc-Vec). Of interest, expression of type I IFN-α receptor (IFNAR) was higher in Panc-PAUF cells than in Panc-Vec cells. Increased expression of IFNAR in turn increased the activation of Stat1 and Tyk2 in Panc-PAUF cells compared with that in Panc-Vec cells. Suppression of Tyk2 and Stat1, which are important downstream molecules for IFN-α signaling, sensitized pancreatic cancer cells to parvovirus H-1-mediated apoptosis. Further, constitutive suppression of PAUF sensitized Bxpc3 pancreatic cancer cells to parvovirus H-1 infection. Taken together, these results suggested that PAUF conferred resistance to pancreatic cancer cells against oncolytic parvovirus H-1 infection through IFNAR-mediated signaling. - Highlights: • PAUF confers resistance against oncolytic parvovirus H-1 infection. • PAUF enhances the expression of IFNAR in Panc-1 cells. • Increased activation of Tyk2 or Stat1 by PAUF provides resistance to parvovirus H-1-mediated apoptosis. • Constitutive inhibition of PAUF enhances parvovirus H-1-mediated oncolysis of Bxpc3 pancreatic cancer cells.

  5. Human GRK4γ142V Variant Promotes Angiotensin II Type I Receptor-Mediated Hypertension via Renal Histone Deacetylase Type 1 Inhibition.

    Science.gov (United States)

    Wang, Zheng; Zeng, Chunyu; Villar, Van Anthony M; Chen, Shi-You; Konkalmatt, Prasad; Wang, Xiaoyan; Asico, Laureano D; Jones, John E; Yang, Yu; Sanada, Hironobu; Felder, Robin A; Eisner, Gilbert M; Weir, Matthew R; Armando, Ines; Jose, Pedro A

    2016-02-01

    The influence of a single gene on the pathogenesis of essential hypertension may be difficult to ascertain, unless the gene interacts with other genes that are germane to blood pressure regulation. G-protein-coupled receptor kinase type 4 (GRK4) is one such gene. We have reported that the expression of its variant hGRK4γ(142V) in mice results in hypertension because of impaired dopamine D1 receptor. Signaling through dopamine D1 receptor and angiotensin II type I receptor (AT1R) reciprocally modulates renal sodium excretion and blood pressure. Here, we demonstrate the ability of the hGRK4γ(142V) to increase the expression and activity of the AT1R. We show that hGRK4γ(142V) phosphorylates histone deacetylase type 1 and promotes its nuclear export to the cytoplasm, resulting in increased AT1R expression and greater pressor response to angiotensin II. AT1R blockade and the deletion of the Agtr1a gene normalize the hypertension in hGRK4γ(142V) mice. These findings illustrate the unique role of GRK4 by targeting receptors with opposite physiological activity for the same goal of maintaining blood pressure homeostasis, and thus making the GRK4 a relevant therapeutic target to control blood pressure.

  6. MAS receptors mediate vasoprotective and atheroprotective effects of candesartan upon the recovery of vascular angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axis functionality.

    Science.gov (United States)

    Pernomian, Larissa; do Prado, Alejandro F; Gomes, Mayara S; Pernomian, Laena; da Silva, Carlos H T P; Gerlach, Raquel F; de Oliveira, Ana M

    2015-10-05

    AT1 antagonists effectively prevent atherosclerosis since AT1 upregulation and angiotensin II-induced proinflammatory actions are critical to atherogenesis. Despite the classic mechanisms underlying the vasoprotective and atheroprotective actions of AT1 antagonists, the cross-talk between angiotensin-converting enzyme-angiotensin II-AT1 and angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axes suggests other mechanisms beyond AT1 blockage in such effects. For instance, angiotensin-converting enzyme 2 activity is inhibited by reactive oxygen species derived from AT1-mediated proinflammatory signaling. Since angiotensin-(1-7) promotes antiatherogenic effects, we hypothesized that the vasoprotective and atheroprotective effects of AT1 antagonists could result from their inhibitory effects on the AT1-mediated negative modulation of vascular angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axis functionality. Interestingly, our results showed that early atherosclerosis triggered in thoracic aorta from high cholesterol fed-Apolipoprotein E-deficient mice impairs angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axis functionality by a proinflammatory-redox AT1-mediated pathway. In such mechanism, AT1 activation leads to the aortic release of tumor necrosis factor-α, which stimulates NAD(P)H oxidase/Nox1-driven generation of superoxide and hydrogen peroxide. While hydrogen peroxide inhibits angiotensin-converting enzyme 2 activity, superoxide impairs MAS functionality. Candesartan treatment restored the functionality of angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axis by inhibiting the proinflammatory-redox AT1-mediated mechanism. Candesartan also promoted vasoprotective and atheroprotective effects that were mediated by MAS since A779 (MAS antagonist) co-treatment inhibited them. The role of MAS receptors as the final mediators of the vasoprotective and atheroprotective effects of candesartan was supported by the vascular actions of angiotensin

  7. Functional analysis of chimeric lysin motif domain receptors mediating Nod factor-induced defense signaling in Arabidopsis thaliana and chitin-induced nodulation signaling in Lotus japonicus.

    Science.gov (United States)

    Wang, Wei; Xie, Zhi-Ping; Staehelin, Christian

    2014-04-01

    The expression of chimeric receptors in plants is a way to activate specific signaling pathways by corresponding signal molecules. Defense signaling induced by chitin from pathogens and nodulation signaling of legumes induced by rhizobial Nod factors (NFs) depend on receptors with extracellular lysin motif (LysM) domains. Here, we constructed chimeras by replacing the ectodomain of chitin elicitor receptor kinase 1 (AtCERK1) of Arabidopsis thaliana with ectodomains of NF receptors of Lotus japonicus (LjNFR1 and LjNFR5). The hybrid constructs, named LjNFR1-AtCERK1 and LjNFR5-AtCERK1, were expressed in cerk1-2, an A. thaliana CERK1 mutant lacking chitin-induced defense signaling. When treated with NFs from Rhizobium sp. NGR234, cerk1-2 expressing both chimeras accumulated reactive oxygen species, expressed chitin-responsive defense genes and showed increased resistance to Fusarium oxysporum. In contrast, expression of a single chimera showed no effects. Likewise, the ectodomains of LjNFR1 and LjNFR5 were replaced by those of OsCERK1 (Oryza sativa chitin elicitor receptor kinase 1) and OsCEBiP (O. sativa chitin elicitor-binding protein), respectively. The chimeras, named OsCERK1-LjNFR1 and OsCEBiP-LjNFR5, were expressed in L. japonicus NF receptor mutants (nfr1-1; nfr5-2) carrying a GUS (β-glucuronidase) gene under the control of the NIN (nodule inception) promoter. Upon chitin treatment, GUS activation reflecting nodulation signaling was observed in the roots of NF receptor mutants expressing both chimeras, whereas a single construct was not sufficient for activation. Hence, replacement of ectodomains in LysM domain receptors provides a way to specifically trigger NF-induced defense signaling in non-legumes and chitin-induced nodulation signaling in legumes. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  8. Orbitofrontal cortex 5-HT2A receptor mediates chronic stress-induced depressive-like behaviors and alterations of spine density and Kalirin7.

    Science.gov (United States)

    Xu, Chang; Ma, Xin-Ming; Chen, Hui-Bin; Zhou, Meng-He; Qiao, Hui; An, Shu-Cheng

    2016-10-01

    Neuroimaging studies show that patients with major depression have reduced volume of the orbitofrontal cortex (OFC). Although the serotonin (5-HT) 2A receptor, which is abundant in the OFC, has been implicated in depression, the underlying mechanisms in the development of stress-induced depression remain unclear. Kalirin-7 (Kal7) is an essential component of mature excitatory sy